Qiaamp Viral Rna Mini Handbook

January 2020
QIAamp® Viral RNA

Mini Handbook
For purification of viral RNA
from plasma, serum, cell-free
body fluids and culture
supernatants
Sample to Insight__
Contents
Kit Contents ............................................................................................................... 4
Storage ..................................................................................................................... 4
Intended Use .............................................................................................................. 5
Safety Information ....................................................................................................... 5
Quality Control........................................................................................................... 6
Introduction ................................................................................................................ 7
Principle .......................................................................................................... 7
Procedure ........................................................................................................ 8
Cellular DNA contamination ............................................................................ 10
Warnings and precautions .............................................................................. 10
Sample volumes ............................................................................................. 11
Lysis .............................................................................................................. 11
Carrier RNA .................................................................................................. 12
Addition of internal controls ............................................................................. 12
Spin and vacuum procedures ........................................................................... 13
Automated purification of RNA on QIAcube Instruments ...................................... 13
Determination of yield ..................................................................................... 14
Equipment and Reagents to Be Supplied by User .......................................................... 15
Important Notes ........................................................................................................ 16
Preparation of reagents ................................................................................... 17
Handling of QIAamp Mini columns .................................................................. 20
Handling guidelines for the QIAvac 24 Plus ...................................................... 22
2 QIAamp Viral RNA Mini Handbook 01/2020

Centrifugation ................................................................................................ 26
Protocol: Purification of Viral RNA (Spin Protocol) ......................................................... 27
Protocol: Purification of Viral RNA (Vacuum Protocol) .................................................... 31
Protocol: Sample Concentration .................................................................................. 36
Protocol: Purification of Cellular, Bacterial or Viral DNA from Urine ................................ 37
Troubleshooting Guide .............................................................................................. 38
Appendix A: General Remarks on Handling RNA......................................................... 41
Ordering Information ................................................................................................ 44
Document Revision History ......................................................................................... 47
QIAamp Viral RNA Mini Handbook 01/2020 3

Kit Contents
QIAamp Viral RNA Mini Kit (50) (250)
Catalog no. 52904 52906
No. of preps 50 250
QIAamp Mini Spin Columns 50 250
Collection Tubes (2 ml) 150 750
Buffer AVL* 31 ml 5 x 31 ml
Buffer AW1* (concentrate) 19 ml 98 ml

†
Buffer AW2 (concentrate) 13 ml 66 ml
Buffer AVE† 3 x 2 ml 10 x 2 ml
Carrier RNA (poly A) 310 µg 5 x 310 µg
* Contains chaotropic salt which is an irritant. Not compatible with disinfecting reagents that contain bleach. See
page 5 for safety information.
†
Contains sodium azide as a preservative.
Storage
QIAamp Mini spin columns should be stored dry at room temperature (15–25°C); storage at
higher temperatures should be avoided. All solutions should be stored at room temperature
unless otherwise stated. QIAamp Mini spin columns and all buffers and reagents can be stored
under these conditions until the expiration date on the kit box without showing any reduction
in performance.

Intended Use
The QIAamp Viral RNA Mini Kit is intended for molecular biology applications. This product
is not intended for the diagnosis, prevention or treatment of a disease.
QIAcube® Connect is designed to perform fully automated purification of nucleic acids and
proteins in molecular biology applications. The system is intended for use by professional users
trained in molecular biological techniques and the operation of QIAcube Connect.
All due care and attention should be exercised in the handling of the products. We recommend
all users of QIAGEN® products to adhere to the NIH guidelines that have been developed for
recombinant DNA experiments or to other applicable guidelines.
Safety Information
When working with chemicals, always wear a suitable lab coat, disposable gloves and
protective goggles. For more information, please consult the appropriate safety data sheets
(SDSs). These are available online in convenient and compact PDF format at
www.qiagen.com/safety where you can find, view and print the SDS for each QIAGEN kit
and kit component.
CAUTION DO NOT add bleach or acidic solutions directly to the sample

preparation waste
Buffers AVL and AW1 contain guanidine salts, which can form highly reactive compounds
when combined with bleach. If liquid containing these buffers is spilt, clean with a suitable
laboratory detergent and water. If the spilt liquid contains potentially infectious agents, clean

the affected area first with laboratory detergent and water, and then with 1% (v/v) sodium
hypochlorite.
Quality Control
In accordance with QIAGEN’s ISO-certified Quality Management System, each lot of QIAamp
Viral RNA Mini Kit is tested against predetermined specifications to ensure consistent product
quality.

Introduction
Please take a few moments to read this handbook carefully before beginning your preparation.
The “Important Notes“, on page 16, and the comments within the QIAamp Viral RNA Mini
protocols, beginning on page 27, are particularly valuable.
Principle
QIAamp Viral RNA Mini Kits provide the fastest and easiest way to purify viral RNA for reliable
use in amplification technologies. Viral RNA can be purified from plasma (treated with
anticoagulants other than heparin), serum and other cell-free body fluids. Samples may be
fresh or frozen, but if frozen, should not be thawed more than once. Repeated freeze–thawing
of plasma samples will lead to reduced viral titers and should be avoided for optimal
sensitivity. Cryoprecipitates accumulate when samples are subjected to repeated freeze–thaw
cycles. This may lead to clogging of the QIAamp membrane when using the vacuum protocol.
QIAamp Viral RNA Mini Kits are for general use and can be used for isolation of viral RNA
from a wide variety of viruses, but performance cannot be guaranteed for every virus.

Procedure
QIAamp Viral RNA Mini Kits represent a well-established technology for general-use viral RNA
preparation. The kit combines the selective binding properties of a silica-based membrane
with the speed of microspin or vacuum technology and is highly suited for simultaneous
processing of multiple samples. QIAamp Viral RNA spin protocols can be fully automated on
QIAcube Connect (see page 13). The sample is first lysed under highly denaturing conditions
to inactivate RNases and to ensure isolation of intact viral RNA. Buffering conditions are then
adjusted to provide optimum binding of the RNA to the QIAamp membrane, and the sample
is loaded onto the QIAamp Mini spin column. The RNA binds to the membrane, and
contaminants are efficiently washed away in two steps using two different wash buffers. High-
quality RNA is eluted in a special RNase-free buffer, ready for direct use or safe storage. The
purified RNA is free of protein, nucleases and other contaminants and inhibitors. The special
QIAamp membrane guarantees extremely high recovery of pure, intact RNA in just 20 minutes
without the use of phenol/chloroform extraction or alcohol precipitation.
Adsorption to the QIAamp membrane
The buffering conditions of the lysate must be adjusted to provide optimum binding conditions
for the viral RNA before loading the sample onto the QIAamp Mini column. Viral RNA is
adsorbed onto the QIAamp silica membrane during two brief centrifugation steps or by
vacuum. Salt and pH conditions in the lysate ensure that protein and other contaminants, which
can inhibit downstream enzymatic reactions, are not retained on the QIAamp membrane. If
the initial sample volume is larger than 140 µl, it will be necessary to load the lysate onto the
QIAamp Mini column in several steps.

Removal of residual contaminants
Viral RNA, bound to the QIAamp membrane, is washed free of contaminants during two short
centrifugation or vacuum steps. The use of two different wash buffers, AW1 and AW2,
significantly improves the purity of the eluted RNA. Optimized wash conditions ensure
complete removal of any residual contaminants without affecting RNA binding.
Elution with Buffer AVE
Buffer AVE is RNase-free water that contains 0.04% sodium azide to prevent microbial growth
and subsequent contamination with RNases. Sodium azide affects spectrophotometric
absorbance readings between 220 and 280 nm but has no effect on downstream
applications, such as RT-PCR. Should you wish to determine the purity of the eluted RNA,
elution with RNase-free water instead of Buffer AVE is recommended.
Cellular DNA contamination
The QIAamp Viral RNA Mini Kit is not designed to separate viral RNA from cellular DNA, and
both will be purified in parallel if present in the sample. To avoid co-purification of cellular
DNA, the use of cell-free body fluids for preparation of viral RNA is recommended. Samples
containing cells, such as cerebrospinal fluid, bone marrow, urine and most swabs, should first
be filtered, or centrifuged for 10 minutes at 1500 x g and the supernatant used. If RNA and
DNA have been isolated in parallel, the eluate can be DNase digested using RNase-free
DNase, followed by heat treatment (15 min, 70°C) to inactivate the DNase.
Warnings and precautions
RNA is extremely sensitive to RNases and should always be prepared with due care. Hands
and dust particles may carry bacteria and molds and are the most common sources of RNase
contamination. Please read “Appendix A: General Remarks on Handling RNA” (page 41)
before starting.

PCR should always be carried out using good laboratory practices. Accordingly, a PCR
laboratory should always be divided into three areas: an area for preparation of reagents, an
area for preparation of samples and an area for amplification and detection. Due to the high
sensitivity of PCR, it is absolutely necessary that all reagents remain pure and uncontaminated,
and should be monitored carefully and routinely. Contaminated reagents must be discarded.
Sample volumes
QIAamp Mini columns can bind RNA greater than 200 nucleotides in length. Actual yield will
depend on sample size, sample storage and virus titer. The procedure is optimized for use
with 140 µl samples, but samples up to 280 µl can be used. Small samples should be adjusted
to 140 µl with phosphate-buffered saline (PBS) before loading, and samples with a low viral
titer should be concentrated to 140 µl before processing. For samples larger than 140 µl, the
amount of lysis buffer and other reagents added to the sample before loading must be
increased proportionally, but the amounts of Buffers AW1 and AW2 used in the wash steps
usually do not need to be increased. If the initial sample volume is increased, application of
the lysed sample to the QIAamp Mini column will require multiple loading steps. There is no
danger of overloading the QIAamp Mini column, and the quality of the purified RNA will be
unaffected. For volumes greater than 560 µl, concentration of the sample is recommended.
See “Protocol: Sample Concentration”, page 36.
Lysis
The sample is first lysed under the highly denaturing conditions provided by Buffer AVL to
inactivate RNases and to ensure isolation of intact viral RNA. Carrier RNA, added to Buffer
AVL, improves the binding of viral RNA to the QIAamp membrane especially in the case of
low-titer samples, and limits possible degradation of the viral RNA due to any residual RNase
activity.

Carrier RNA
Carrier RNA serves two purposes. Firstly, it enhances binding of viral nucleic acids to the
QIAamp Mini membrane, especially if there are very few target molecules in the sample.
Secondly, the addition of large amounts of carrier RNA reduces the chance of viral RNA
degradation in the rare event that RNase molecules escape denaturation by the chaotropic
salts and detergent in Buffer AVL. If carrier RNA is not added to Buffer AVL this may lead to
reduced viral RNA recovery.
The amount of lyophilized carrier RNA provided is sufficient for the volume of Buffer AVL
supplied with the kit. The concentration of carrier RNA has been adjusted so that the QIAamp
Viral RNA Mini Kit can be used as a generic purification system compatible with many different
amplification systems and is suitable for a wide range of RNA viruses.
Different amplification systems vary in efficiency depending on the total amount of nucleic acid
present in the reaction. Eluates from this kit contain both viral nucleic acids and carrier RNA, and
amounts of carrier RNA will greatly exceed amounts of viral nucleic acids. Calculations of how
much eluate to add to downstream amplifications should therefore be based on the amount of
carrier RNA added. To obtain the highest levels of sensitivity in amplification reactions, it may
be necessary to adjust the amount of carrier RNA added to Buffer AVL.
Addition of internal controls
Using the QIAamp Viral RNA Mini protocols in combination with commercially available
amplification systems may require the introduction of an internal control into the purification
procedure. Internal control RNA or DNA should be added together with the carrier RNA to
the lysis buffer. For optimal purification efficiency, internal control molecules should be longer
than 200 nucleotides, as smaller molecules are not efficiently recovered.
Refer to the manufacturer’s instructions to determine the optimal concentration. Using a

concentration other than that recommended may reduce amplification efficiency.

Spin and vacuum procedures
The QIAamp Viral RNA Mini purification procedure is carried out in three steps using QIAamp
Mini columns in a standard microcentrifuge, on a vacuum manifold or on QIAcube Connect
(see page 13). The procedures are designed to ensure that there is no sample-to-sample cross-
contamination and allow safe handling of potentially infectious samples.
QIAamp Mini columns fit into most standard microcentrifuge tubes. In the spin protocol, due
to the volume of filtrate, 2 ml collection tubes (provided) are required to support the QIAamp
Mini column during loading and wash steps. For the vacuum protocol, a vacuum manifold
(QIAvac 24 Plus or equivalent see page 18) and a vacuum pump capable of producing a
vacuum of –800 to –900 mbar (e.g., Vacuum Pump) are required.
Eluted RNA can be collected in standard 1.5 ml microcentrifuge tubes (not provided). These
tubes must be RNase-free to avoid degradation of viral RNA by RNases.
Automated purification of RNA on QIAcube Instruments
Purification of RNA can be fully automated on QIAcube Connect or the classic QIAcube. The
innovative QIAcube instruments use advanced technology to process QIAGEN spin columns,
enabling seamless integration of automated, low-throughput sample prep into your laboratory
workflow. Sample preparation using QIAcube instruments follows the same steps as the
manual procedure (i.e., lyse, bind, wash and elute), enabling you to continue using the
QIAamp Viral RNA Mini Kit for purification of high-quality RNA.
QIAcube instruments are preinstalled with protocols for purification of plasmid DNA, genomic
DNA, RNA, viral nucleic acids and proteins, plus DNA and RNA cleanup. The range of
protocols available is continually expanding, and additional QIAGEN protocols can be
downloaded free of charge at www.qiagen.com/qiacubeprotocols.

QIAcube Connect.
Determination of yield
Yields of viral RNA isolated from biological samples are normally less than 1 µg and therefore
difficult to determine photometrically. Keep in mind that the carrier RNA (5.6 µg per 140 µl
sample) will account for most of the RNA present. Quantitative RT-PCR is recommended for
determination of viral RNA yield.
Determination of viral RNA length
The size distribution of viral RNA purified using QIAamp spin columns can be checked by
denaturing agarose gel electrophoresis followed by hybridization with a virus-specific labeled
probe and autoradiography (Sambrook, J. and Russell, D. W. [2001] Molecular Cloning: A
Laboratory Manual, 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).

Equipment and Reagents to Be Supplied by User
When working with chemicals, always wear a suitable lab coat, disposable gloves and
protective goggles. For more information, consult the appropriate safety data sheets (SDSs),
available from the product supplier.
Ethanol (96–100%)*
1.5 ml microcentrifuge tubes
Sterile, RNase-free pipette tips (pipette tips with aerosol barriers for preventing cross-
contamination are recommended)
Microcentrifuge (with rotor for 1.5 ml and 2 ml tubes)
For vacuum protocols

QIAvac 24 Plus vacuum manifold (cat. no. 19413) or equivalent
VacConnectors (cat. no. 19407)
Vacuum Regulator (cat. no. 19530) for easy monitoring of vacuum pressures and easy
releasing of vacuum
Vacuum Pump (cat. no. 84010 [USA and Canada], 84000 [Japan] or 84020 [rest of
world]) or equivalent pump capable of producing a vacuum of –800 to –900 mbar
Optional: VacValves (cat. no. 19408)
Optional: QIAvac Connecting System (cat. no. 19419)
* Do not use denatured alcohol, which contains other substances, such as methanol or methylethylketone.

Important Notes
If preparing RNA for the first time, see “Appendix A: General Remarks on Handling RNA”,
page 41. All steps of the QIAamp Viral RNA Mini protocols should be performed quickly and
at room temperature (15–25°C).
After collection and centrifugation, plasma (untreated or treated with anticoagulants other than
heparin) or serum can be stored at 2–8°C for up to 6 hours. For long-term storage, freezing
at –30°C to –15°C or –90°C to –65°C in aliquots is recommended. Frozen plasma or serum
samples must not be thawed more than once. Repeated freezing and thawing leads to
denaturation and precipitation of proteins, causing reduced viral titers and subsequently
reduced yields of the isolated viral RNA. In addition, cryoprecipitates formed by freeze–
thawing will cause clogging of the QIAamp membrane. If cryoprecipitates are visible, they
can be pelleted by briefly centrifuging at 6800 x g for 3 minutes. The cleared supernatant
should be removed, without disturbing the pellet, and processed immediately. This step will
not reduce viral titers.
The QIAamp Viral RNA Mini procedure is not designed to separate RNA from DNA. To avoid
cellular DNA contamination follow the guidelines in ”Cellular DNA contamination” on page
10.
The QIAamp Viral RNA Mini procedure isolates all RNA molecules larger than
200 nucleotides. Smaller RNA molecules will not bind quantitatively under the conditions used.

Preparation of reagents
Addition of carrier RNA to Buffer AVL*
Add 310 µl Buffer AVE to the tube containing 310 µg lyophilized carrier RNA to obtain a
solution of 1 µg/µl. Dissolve the carrier RNA thoroughly, divide it into conveniently sized
aliquots, and store it at –30°C to –15°C. Do not freeze–thaw the aliquots of carrier RNA more
than three times.
Check Buffer AVL for precipitate, and if necessary incubate at 80°C until the precipitate is
dissolved. Calculate the volume of Buffer AVL–carrier RNA mix needed per batch of samples
by selecting the number of samples to be simultaneously processed from Table 1 (next page).
For larger numbers of samples, volumes can be calculated using the following sample
calculation:
n x 0.56 ml = y ml
y ml x 10 µl/ml = z µl
Where: n = number of samples to be processed simultaneously
y = calculated volume of Buffer AVL
z = volume of carrier RNA–Buffer AVE to add to Buffer AVL
Gently mix by inverting the tube 10 times. To avoid foaming, do not vortex.
* Contains chaotropic salt. Take appropriate laboratory safety measures and wear gloves when handling. Not
compatible with disinfecting agents that contain bleach. See page 5 for safety information.

Table 1. Volumes of Buffer AVL and carrier RNA–Buffer AVE mix required for the QIAamp Viral RNA Mini procedure
No. samples Buffer AVL (ml) Carrier RNA–AVE (µl) No. samples Buffer AVL (ml) Carrier RNA–AVE (µl)
1 0.56 5.6 13 7.28 72.8
2 1.12 11.2 14 7.84 78.4
3 1.68 16.8 15 8.4 84.0
4 2.24 22.4 16 8.96 89.6
5 2.80 28.0 17 9.52 95.2
6 3.36 33.6 18 10.08 100.8
7 3.92 39.2 19 10.64 106.4
8 4.48 44.8 20 11.20 112.0
9 5.04 50.4 21 11.76 117.6
10 5.60 56.0 22 12.32 123.2
11 6.16 61.6 23 12.88 128.8
12 6.72 67.2 24 13.44 134.4
Note: The sample-preparation procedure is optimized for 5.6 µg of carrier RNA per sample.
If less carrier RNA has been shown to be better for your amplification system, transfer only the
required amount of dissolved carrier RNA to the tubes containing Buffer AVL. (Use of less than
5.6 µg carrier RNA per sample must be validated for each particular sample type and
downstream assay.)
Buffer AVL–carrier RNA should be prepared fresh, and is stable at 2–8°C for up to 48 hours.
This solution develops a precipitate when stored at 2–8°C that must be redissolved by warming
at 80°C before use. Do not warm Buffer AVL–carrier RNA solution more than six times. Do not
incubate at 80°C for more than 5 minutes. Frequent warming and extended incubation will cause
degradation of carrier RNA, leading to reduced recovery of viral RNA and eventually false
negative RT-PCR results. This is particularly the case with low-titer samples.

Buffer AW1*
Buffer AW1 is supplied as a concentrate. Before using for the first time, add the appropriate
amount of ethanol (96–100%) as indicated on the bottle and in Table 2. Buffer AW1 is stable
for 1 year when stored closed at room temperature (15–25°C), but only until the kit expiration
date.
Table 2. Preparation of Buffer AW1
Kit cat. no. No. preps AW1 concentrate Ethanol Final volume
52904 50 19 ml 25 ml 44 ml
52904 250 98 ml 130 ml 228 ml
Buffer AW2†
Buffer AW2 is supplied as a concentrate. Before using for the first time, add the appropriate
amount of ethanol (96–100%) to Buffer AW2 concentrate as indicated on the bottle and in
Table 3.
Buffer AW2 is stable for 1 year when stored closed at room temperature (15–25°C), but only
until the kit expiration date.
Table 3. Preparation of Buffer AW2
Kit cat. no. No. preps AW2 concentrate Ethanol Final volume
52904 50 13 ml 30 ml 43 ml
52904 250 66 ml 160 ml 226 ml
* Contains chaotropic salt. Take appropriate laboratory safety measures and wear gloves when handling. Not
compatible with disinfecting agents that contain bleach. See page 5 for safety information.
† Contains sodium azide as a preservative.

Handling of QIAamp Mini columns
Owing to the sensitivity of nucleic acid amplification technologies, the following precautions
are necessary when handling QIAamp Mini columns to avoid cross-contamination between
sample preparations:
Carefully apply the sample or solution to the QIAamp Mini column. Pipet the sample into
the QIAamp Mini column without wetting the rim of the column.
Change pipette tips between all liquid transfer steps. The use of aerosol-barrier tips is
recommended.
Avoid touching the QIAamp membrane with the pipette tip.
After all pulse-vortexing steps, briefly centrifuge 1.5 ml microcentrifuge tubes to remove
drops from the inside of the lid.
Wear gloves throughout the procedure. In case of contact between gloves and sample,
change gloves immediately.
Spin protocol
Close the QIAamp Mini column before placing it in the microcentrifuge. Centrifuge as
described.
Remove the QIAamp Mini column and collection tube from the microcentrifuge. Place the
QIAamp Mini column in a new collection tube. Discard the filtrate and the old collection
tube. Please note that the filtrate may contain hazardous waste and should be disposed
of properly.
Open only one QIAamp Mini column at a time, and take care to avoid generating aerosols.
For efficient parallel processing of multiple samples, it is recommended to fill a rack with
collection tubes to which the QIAamp Mini columns can be transferred after centrifugation.
Used collection tubes containing the filtrate can be discarded, and the new collection tubes
containing the QIAamp Mini columns can be placed directly in the microcentrifuge.

Vacuum protocol on the QIAvac 24 Plus
The QIAvac 24 Plus is designed for fast and efficient vacuum processing of up to 24 QIAGEN
spin columns in parallel. Samples and wash solutions are drawn through the column
membranes by vacuum instead of centrifugation, providing greater speed and reduced hands-
on time in purification procedures.
In combination with the QIAvac Connecting System (optional), the QIAvac 24 Plus can be
used as a flow-through system. The sample flow-through is collected in a separate waste bottle.
For maintenance of the QIAvac 24 Plus, please refer to the handling guidelines in the QIAvac
24 Plus Handbook.
Processing QIAamp Mini columns on the QIAvac 24 Plus
QIAamp Mini columns are processed on the QIAvac 24 Plus using VacConnectors and
reusable VacValves. VacValves (optional) are inserted directly into the luer slots of the QIAvac
24 Plus manifold and ensure a steady flow rate, facilitating parallel processing of samples of
different natures (e.g., plasma and serum), volumes or viscosities. They should be used if
sample flow rates differ significantly to ensure consistent vacuum. VacConnectors are
disposable connectors that fit between QIAamp Mini columns and VacValves or between
QIAamp Mini columns and the QIAvac 24 Plus manifold. They prevent direct contact between
the column and VacValve or QIAvac 24 Plus manifold during purification, thereby avoiding
any cross-contamination between samples. VacConnectors are discarded after a single use.

Handling guidelines for the QIAvac 24 Plus
Always place the QIAvac 24 Plus on a secure bench top or work area. If dropped, the
QIAvac 24 Plus manifold may crack.
Always store the QIAvac 24 Plus clean and dry. For cleaning procedures, see the
QIAvac 24 Plus Handbook.
The components of the QIAvac 24 Plus are not resistant to certain solvents (Table 4, next
page). If these solvents are spilled on the unit, rinse it thoroughly with water.
To ensure consistent performance, do not apply silicone or vacuum grease to any part of
the QIAvac 24 Plus manifold.
Always use caution and wear safety glasses when working near a vacuum manifold
under pressure.
Contact QIAGEN Technical Services or your local distributor for information concerning
spare or replacement parts.
The vacuum pressure is the pressure differential between the inside of the vacuum manifold
and the atmosphere (standard atmospheric pressure 1013 millibar or 760 mm Hg) and
can be measured using the QIAvac Connecting System or a vacuum regulator (Figure 1,
next page). The vacuum protocol requires a vacuum pump capable of producing a vacuum
of –800 to –900 mbar (e.g., QIAGEN's, Vacuum Pump). Higher vacuum pressures must be
avoided. Use of vacuum pressures lower than recommended may reduce DNA yield and
purity and increase the frequency of clogged membranes.

Table 4. Chemical resistance properties of the QIAvac 24 Plus
Resistant to: Not resistant to:
Acetic acid Benzene
Chromic acid Chloroform
Chaotropic salts Ethers
Chlorine bleach Phenol
Hydrochloric acid Toluene
SDS –
Sodium chloride –
Sodium hydroxide –
®
Tween 20 –
Urea –
Regulator gauge
Knob
Figure 1. The Vacuum Regulator.

Setup of the QIAvac 24 Plus vacuum manifold
1. Connect the QIAvac 24 Plus to a vacuum source. If using the QIAvac Connecting
System, connect the system to the manifold and vacuum source as described in
Appendix A of the QIAvac 24 Plus Handbook.
Recommended: Insert a VacValve into each luer slot of the QIAvac 24 Plus that is to be
used (Figure 2, next page).
VacValves should be used if flow rates of samples differ significantly to ensure consistent
vacuum.
2. Insert a VacConnector into each VacValve (Figure 2) or directly into each luer slot of the
QIAvac 24 Plus that is to be used. Close unused luer slots with luer plugs or close the
inserted VacValve.
Perform this step directly before starting the purification to avoid exposure of
VacConnectors to potential contaminants in the air.
3. Place the QIAamp Mini columns into the VacConnectors on the manifold (Figure 2).
4. For nucleic acid purification, follow the instructions in the vacuum protocol. Discard the
VacConnectors appropriately after use.
Leave the lid of the QIAamp Mini column open while applying vacuum.
Switch off the vacuum between steps to ensure that a consistent, even vacuum is applied
during processing. For faster vacuum release, a vacuum regulator should be used (Figure 1).
Note: Each VacValve can be closed individually when the sample is completely drawn
through the spin column, allowing parallel processing of samples of different volumes or
viscosities.
5. After processing samples, clean the QIAvac 24 Plus (see “Cleaning and
Decontaminating the QIAvac 24 Plus” in the QIAvac 24 Plus Handbook).
Note: Buffers AVL and AW1 used in QIAamp Viral RNA Mini procedure are not
compatible with disinfecting agents containing bleach. See page 5 for safety
information.

5
Figure 2. Setting up the QIAvac 24 Plus with QIAamp Mini columns using VacValves and VacConnectors. 1 = QIAvac
24 Plus vacuum manifold; 2 = Luer slot of the QIAvac 24 Plus; 3 = VacValve (optional; must be purchased separately);
4 = VacConnector (must be purchased separately); 5 = QIAamp column; 6 = Luer slot closed with luer plug.

Centrifugation
QIAamp Mini columns will fit into most standard 1.5 ml or 2 ml microcentrifuge tubes.
Additional 2 ml collection tubes are available separately.
Centrifugation of QIAamp Mini columns is performed at 6000 x g (8000 rpm) to limit

centrifuge noise. Centrifugation at full speed will not affect RNA yield. Centrifugation at lower
speeds for lysate loading and the first wash step is also acceptable, provided that the complete
solution is transferred through the membrane. At the second wash step centrifugation at full
speed is strongly recommended.
All centrifugation steps are carried out at room temperature (15–25°C).

Protocol: Purification of Viral RNA (Spin Protocol)
This protocol is for purification of viral RNA from 140 µl plasma, serum, urine, cell-culture
media or cell-free body fluids using a microcentrifuge. For automated purification of viral RNA
using the QIAamp Viral RNA Mini Kit on QIAcube Connect, refer to the QIAcube Connect
User Manual and the relevant protocol sheet.
Larger starting volumes, up to 560 µl (in multiples of 140 µl), can be processed by increasing the
initial volumes proportionally and loading the QIAamp Mini column multiple times, as described
below in the protocol. Some samples with very low viral titers should be concentrated before the
purification procedure; see ”Protocol: Sample Concentration“ (page 36).
Alternatively, larger sample volumes can be processed using one of the following kits, which
provide simultaneous purification of viral DNA and RNA:
QIAamp MinElute® Spin Kit* – 200 µl

QIAamp MinElute Vacuum Kit – 500 µl
QIAamp UltraSens® Virus Kit – 1000 µl
Important points before starting
Read “Important Notes” (page 16) before starting the protocol.

* Fully automatable on QIAcube Connect. See www.qiagen.com/qiacubeprotocols for protocols.

Things to do before starting
Equilibrate samples to room temperature (15–25°C).
Equilibrate Buffer AVE to room temperature for elution in step 11.
Check that Buffer AW1 and Buffer AW2 have been prepared according to the
instructions on page 19.
Add carrier RNA reconstituted in Buffer AVE to Buffer AVL according to instructions on
page 17.
Procedure
1. Pipet 560 µl prepared Buffer AVL containing carrier RNA into a 1.5 ml microcentrifuge
tube.
Note: If the sample volume is larger than 140 µl, increase the amount of Buffer AVL–carrier
RNA proportionally (e.g., a 280 µl sample will require 1120 µl Buffer AVL–carrier RNA)
and use a larger tube.
2. Add 140 µl plasma, serum, urine, cell-culture supernatant or cell-free body fluid to the
Buffer AVL–carrier RNA in the microcentrifuge tube. Mix by pulse-vortexing for 15 s.
Note: To ensure efficient lysis, it is essential that the sample is mixed thoroughly with
Buffer AVL to yield a homogeneous solution. Frozen samples that have only been thawed
once can also be used.
3. Incubate at room temperature (15–25°C) for 10 min.
Note: Viral particle lysis is complete after lysis for 10 min at room temperature. Longer
incubation times have no effect on the yield or quality of the purified RNA.
4. Briefly centrifuge the tube to remove drops from the inside of the lid.

5. Add 560 µl ethanol (96–100%) to the sample, and mix by pulse-vortexing for 15 s. After
mixing, briefly centrifuge the tube to remove drops from inside the lid.
Note: Use only ethanol, since other alcohols may result in reduced RNA yield and purity.
Do not use denatured alcohol, which contains other substances, such as methanol or
methylethylketone. If the sample volume is greater than 140 µl, increase the amount of
ethanol proportionally (e.g., a 280 µl sample will require 1120 µl ethanol). To ensure
efficient binding, it is essential that the sample is mixed thoroughly with the ethanol to
yield a homogeneous solution.
6. Carefully apply 630 µl of the solution from step 5 to the QIAamp Mini column (in a 2 ml
collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g
(8000 rpm) for 1 min. Place the QIAamp Mini column into a clean 2 ml collection tube,
and discard the tube containing the filtrate.
Note: Close each spin column to avoid cross-contamination during centrifugation.
Note: Centrifugation is performed at 6000 x g (8000 rpm) to limit microcentrifuge noise.
Centrifugation at full speed will not affect the yield or purity of the viral RNA. If the
solution has not completely passed through the membrane, centrifuge again at a higher
speed until all of the solution has passed through.
7. Carefully open the QIAamp Mini column, and repeat step 6. If the sample volume was
greater than 140 µl, repeat this step until all of the lysate has been loaded onto the spin
column.
8. Carefully open the QIAamp Mini column, and add 500 µl Buffer AW1. Close the cap,
and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini column in a
clean 2 ml collection tube (provided), and discard the tube containing the filtrate.
Note: It is not necessary to increase the volume of Buffer AW1 even if the original
sample volume was larger than 140 µl.

9. Carefully open the QIAamp Mini column, and add 500 µl Buffer AW2. Close the cap
and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min. Continue directly with
step 11, or to eliminate possible Buffer AW2 carryover, perform step 10 and then
continue with step 11.
Note: Residual Buffer AW2 in the eluate may cause problems in downstream
applications. Some centrifuge rotors may vibrate upon deceleration, resulting in flow-
through, containing Buffer AW2, contacting the QIAamp Mini column.
Removing the QIAamp Mini column and collection tube from the rotor may also cause
flow-through to come into contact with the QIAamp Mini column. In these cases, the
optional step 10 should be performed.
Recommended: Place the QIAamp Mini column in a new 2 ml collection tube (not
provided), and discard the old collection tube with the filtrate. Centrifuge at full speed for
1 min.
10. Place the QIAamp Mini column in a clean 1.5 ml microcentrifuge tube (not provided).
Discard the old collection tube containing the filtrate. Carefully open the QIAamp Mini
column and add 60 µl Buffer AVE equilibrated to room temperature. Close the cap, and
incubate at room temperature for 1 min.
11. Centrifuge at 6000 x g (8000 rpm) for 1 min.
A single elution with 60 µl Buffer AVE is sufficient to elute at least 90% of the viral RNA
from the QIAamp Mini column. Performing a double elution using 2 x 40 µl Buffer AVE
will increase yield by up to 10%. Elution with volumes of less than 30 µl will lead to
reduced yields and will not increase the final concentration of RNA in the eluate.
Viral RNA is stable for up to one year when stored at –30°C to –15°C or at –90°C to
–65°C.

Protocol: Purification of Viral RNA (Vacuum
Protocol)
This protocol is for purification of viral RNA from 140 µl plasma, serum, urine, cell-culture
media or cell-free body fluids using the QIAvac 24 Plus or equivalent vacuum manifold.
Larger starting volumes, up to 560 µl (in multiples of 140 µl), can be processed by
increasing the initial volumes proportionally and loading the QIAamp Mini spin column
multiple times, as described below in the protocol. Some samples with very low viral titers
should be concentrated before the purification procedure; see ”Protocol: Sample
Concentration“ (page 36).
Alternatively, larger sample volumes can be processed using one of the following kits, which
provide simultaneous purification of viral DNA and RNA:
QIAamp MinElute Spin Kit* – 200 µl

QIAamp MinElute Vacuum Kit – 500 µl
QIAamp UltraSens Virus Kit – 1000 µl
Important points before starting
Read “Important Notes” (page 16) before starting the protocol.

* Fully automatable on QIAcube Connect. See www.qiagen.com/qiacubeprotocols for protocols.

Things to do before starting
Equilibrate samples to room temperature (15–25°C).
Equilibrate Buffer AVE to room temperature for elution in step 10.
Check that Buffer AW1 and Buffer AW2 have been prepared according to the
instructions on page 19.
Add carrier RNA reconstituted in Buffer AVE to Buffer AVL according to instructions on
page 17.
For processing using VacConnectors and VacValves, set up the QIAvac 24 Plus, as
described on page 24.
Procedure
1. Pipet 560 µl prepared Buffer AVL containing carrier RNA into a 1.5 ml microcentrifuge
tube.
If the sample volume is larger than 140 µl, increase the amount of Buffer AVL–carrier
RNA proportionally (e.g., a 280 µl sample will require 1120 µl Buffer AVL–carrier RNA)
and use a larger tube.
2. Add 140 µl plasma, serum, urine, cell-culture supernatant or cell-free body fluid to the
Buffer AVL–carrier RNA in the microcentrifuge tube. Mix by pulse-vortexing for 15 s.
To ensure efficient lysis, it is essential that the sample is mixed thoroughly with Buffer AVL
to yield a homogeneous solution. Frozen samples that have only been thawed once can
also be used.
3. Incubate at room temperature (15–25°C) for 10 min.
Viral particle lysis is complete after lysis for 10 min at room temperature. Longer
incubation times have no effect on the yield or quality of the purified RNA.
4. Briefly centrifuge the tube to remove drops from the inside of the lid.

5. Add 560 µl ethanol (96–100%) to the sample, and mix by pulse-vortexing for 15 s. After
mixing, briefly centrifuge the tube to remove drops from inside the lid.
Insert a QIAamp Mini column into the VacConnector on the QIAvac 24 Plus vacuum
manifold.
Use only ethanol, since other alcohols may result in reduced yield and purity of the RNA.
Do not use denatured alcohol, which contains other substances, such as methanol or
methylethylketone. If the sample volume is greater than 140 µl, increase the amount of
ethanol proportionally (e.g., a 280 µl sample will require 1120 µl ethanol). To ensure
efficient binding, it is essential that the sample is mixed thoroughly with the ethanol to
yield a homogeneous solution.
The collection tube from the blister pack can be saved for the centrifugation in step 14.
6. Make sure that the main vacuum valve (between the vacuum pump and the vacuum
manifold) and the screw cap valve (on the end of the QIAvac 24 Plus vacuum manifold)
are closed. Switch on the vacuum pump by pressing the power switch.
The vacuum is applied only to the connecting system (if used) and not to the vacuum manifold.
Note: For fast and convenient release of the vacuum pressure, the QIAvac Connecting
System or the Vacuum Regulator should be used.
7. Carefully apply 630 µl of the lysate from step 5 into the QIAamp Mini column without
wetting the rim. Avoid touching the QIAamp Mini column membrane with the pipette tip.

8. Open the main vacuum valve. Be sure to leave the lid of the QIAamp Mini column open
while applying vacuum. After all lysates have been drawn through the QIAamp Mini
column, close the main vacuum valve and open the screw cap valve to vent the manifold.
Close the screw cap valve after the vacuum is released from the manifold.
After closing the main vacuum valve, the vacuum is applied only to the connecting system
(if used) and not the vacuum manifold.
Note: If the lysates from individual samples have not completely passed through the
membrane despite the VacValves of all other QIAamp Mini columns being closed, place
the QIAamp Mini column into a clean 2 ml collection tube (not provided), close the cap,
and centrifuge at full speed for 3 min or until it has completely passed through. Continue
with step 7 of the spin protocol (page 27). Additional collection tubes can be purchased
separately.
Centrifugation is performed at 6000 x g (8000 rpm) to limit microcentrifuge noise.
Centrifugation at full speed will not affect the yield or purity of viral RNA.
9. Repeat steps 7 and 8.
If the sample volume was greater than 140 µl, repeat these steps until all of the lysate
has been drawn through the QIAamp Mini column.
10. Apply 750 µl Buffer AW1 to the QIAamp Mini column without wetting the rim.
Avoid touching the QIAamp Mini column membrane with the pipette tip.
It is not necessary to increase the volume of Buffer AW1 even if the original sample
volume was larger than 140 µl.
11. Open the main vacuum valve. After all Buffer AW1 has been drawn through the
QIAamp Mini column, close the main vacuum valve and open the screw cap valve to
vent the manifold. Close the screw cap valve after the vacuum is released from the
manifold.
12. Apply 750 µl Buffer AW2 to the QIAamp Mini column without wetting the rim. Avoid
touching the QIAamp Mini column membrane with the pipette tip. Leave the lid of the
column open.

13. Open the main vacuum valve. After all Buffer AW2 has been drawn through the
QIAamp Mini column, close the main vacuum valve and open the screw cap valve to
vent the manifold. Close the screw cap valve after the vacuum is released from the
manifold.
14. Close the lid of the QIAamp Mini column. Remove it from the vacuum manifold and
discard the VacConnector. Place the QIAamp Mini spin column in a clean 2 ml collection
tube saved from step 5, and centrifuge at full speed for 1 min to dry the membrane
completely.
15. Place the QIAamp Mini spin column into a clean 1.5 ml microcentrifuge tube (not
provided). Discard the collection tube containing the filtrate. Carefully open the QIAamp
Mini spin column. Add 60 µl Buffer AVE equilibrated to room temperature. Close the
cap, and incubate at room temperature for 1 min. Centrifuge at 6000 x g (8000 rpm)
for 1 min.
A single elution with 60 µl Buffer AVE is sufficient to elute at least 90% of the viral RNA
from the QIAamp Mini spin column. Performing a double elution using 2 x 40 µl Buffer
AVE will increase yield by up to 10%. Elution with volumes of less than 30 µl will lead to
reduced yields and will not increase the final concentration of RNA in the eluate.
Viral RNA is stable for up to one year when stored at –30°C to –15°C or at –90°C to
–65°C.

Protocol: Sample Concentration
Plasma, serum, urine, cerebrospinal fluid, bone marrow and other body fluids often have very
low viral titers. In these cases, concentrating samples of up to 3.5 ml to a final volume of
140 µl is recommended.
Important point before starting
Use centrifugal microconcentrators, such as Centricon® 100 (Amicon: 2 ml, cat. no.
4211), Microsep 100 (Filtron: 3.5 ml, cat. no. OD100C40), Ultrafree® CL (Millipore:
2 ml, cat. no. UFC4 THK 25) or equivalent from other suppliers.
Procedure
Apply up to 3.5 ml sample to the microconcentrator, following the manufacturer’s

instructions.
Centrifuge according to manufacturer’s instructions to a final volume of 140 µl.
Some samples, plasma in particular, may be difficult to concentrate to 140 µl due to high
viscosity. Centrifugation for up to 6 hours may be necessary.
Pipet 140 µl concentrated sample into a 1.5 ml microcentrifuge tube and follow
“Protocol: Purification of Viral RNA (Spin Protocol)”, page 27.

Protocol: Purification of Cellular, Bacterial or Viral
DNA from Urine
Buffer AVL, used in the QIAamp Viral RNA Mini procedure, inactivates the numerous
unidentified PCR inhibitors found in urine. For isolation of cellular, bacterial or viral DNA from
urine for use in PCR, “Protocol: Purification of Viral RNA (Spin Protocol)”, page 27, is
recommended.
Urine often contains very low numbers of cells, bacteria or viruses. In these cases, we
recommend concentrating samples of up to 3.5 ml to a final volume of 140 µl, as described
below, before processing.
For purification of DNA from Gram-positive bacteria, please contact QIAGEN Technical
Services.
Important point before starting
Use centrifugal microconcentrators, such as Centricon-100 (Amicon: 2 ml, cat. no. 4211),
Microsep 100 (Filtron: 3.5 ml, cat. no. OD100C40), Ultrafree-CL (Millipore: 2 ml, cat. no.
UFC4 THK 25) or equivalent from other suppliers.
Procedure
1. Apply up to 3.5 ml of sample to the microconcentrator following the manufacturer’s

instructions.
2. Centrifuge according to manufacturer’s instructions to a final volume of 140 µl.
Some samples, plasma in particular, may be difficult to concentrate to 140 µl due to high
viscosity. Centrifugation for up to 6 hours may be necessary.
3. Pipet 140 µl concentrated sample into a 1.5 ml microcentrifuge tube and follow Protocol:
Purification of Viral RNA (Spin Protocol) on page 27.

Troubleshooting Guide
This troubleshooting guide may be helpful in solving any problems that may arise. For more
information, see also the Frequently Asked Questions page at our Technical Support Center:
www.qiagen.com/FAQ/FAQList.aspx. The scientists in QIAGEN Technical Services are
always happy to answer any questions you may have about either the information and/or
protocols in this handbook or sample and assay technologies (for contact information, visit
www.qiagen.com).
Comments and suggestions
Little or no RNA in the eluate
a) Carrier RNA not added to Reconstitute carrier RNA in Buffer AVE and mix with Buffer AVL as described
Buffer AVL on page 17. Repeat the purification procedure with new samples.
b) Degraded carrier RNA Carrier RNA reconstituted in Buffer AVE was not stored at –30°C to –15°C or
underwent multiple freeze–thaw cycles. Alternatively, Buffer AVL–carrier RNA
mixture was stored for more than 48 hours at 2–8°C. Prepare a new tube of
carrier RNA dissolved in Buffer AVE and mix with Buffer AVL. Repeat the
purification procedure with new samples.
c) Sample frozen and thawed Repeated freezing and thawing should be avoided. Always use fresh samples
more than once or samples thawed only once.
d) Low concentration of virus in Concentrate the sample volume to 140 µl using a microconcentrator. Repeat
the sample the RNA purification procedure with a new sample. See “Protocol: Sample
Concentration” on page 36.
e) Inefficient protein Precipitate, formed in Buffer AVL–carrier RNA after storage at 2–8°C, was not
denaturation in Buffer AVL redissolved by heating before starting the procedure. Redissolve the
precipitate and repeat the procedure with a new sample.
f) Buffer AVL prepared Check Buffer AVL for precipitate. Dissolve precipitate by incubation at 80°C.
incorrectly
g) No ethanol added to the Repeat the purification procedure with a new sample.
lysate (step 5)
h) Low percentage ethanol Repeat the purification procedure with a new sample. Use 96–100% ethanol
used in step 5. Do not use denatured alcohol, which contains other substances,
such as methanol or methylethylketone.

i) Isopropanol used instead of We recommend the use of ethanol as isopropanol causes reduced yields.
ethanol
j) RNA degraded Often RNA is degraded by RNases in the starting material (plasma, serum,
body fluids). Ensure that the samples are processed quickly. If necessary, add
RNase inhibitor to the sample. Check for RNase contamination of buffers and
water, and ensure that no RNase is introduced during the procedure.
k) RNase contamination in Discard contaminated Buffer AVE. Repeat the purification procedure with a
Buffer AVE new sample and a fresh tube of Buffer AVE. Additional Buffer AVE is
available separately.
l) Buffer AW1 or AW2 Check that Buffer AW1 and AW2 concentrates were diluted with correct
prepared incorrectly volumes of pure (96–100%) ethanol. Do not use denatured alcohol, which
contains other substances, such as methanol or methylethylketone. Repeat the
purification procedure with a new sample.
m) Buffer AW1 or AW2 Check that Buffer AW1 and AW2 concentrates were diluted with 96–100%
prepared with 70% ethanol ethanol. Repeat the purification procedure with a new sample.
n) Buffers AW1 and AW2 used Ensure that Buffer AW1 and Buffer AW2 are used in used in the correct order
in the wrong order in the protocol. Repeat the purification procedure with a new sample.
RNA does not perform well in subsequent enzymatic reactions
a) Little or no RNA in the Check “Little or no RNA in the eluate,” above, for possible reasons.
eluate
b) Inefficient virus lysis in Buffer Precipitate formed in Buffer AVL–carrier RNA due to temperature change before
AVL start of process. Repeat the procedure with new samples, and ensure that no
precipitate has formed in Buffer AVL–carrier RNA at the beginning of the
process.
c) Buffer AVL prepared Ensure that carrier RNA has been reconstituted in Buffer AVE and added to
incorrectly Buffer AVL (see page 17).
d) Too much carrier RNA in the Determine the maximum amount of carrier RNA suitable for your RT-PCR. Adjust
eluate the concentration of carrier RNA added to Buffer AVL accordingly.
e) Reduced sensitivity Determine the maximum volume of eluate suitable for your RT-PCR. Reduce the
volume of eluate added to the RT-PCR.
f) Buffers AW1 and AW2 used Ensure that Buffer AW1 and Buffer AW2 are used in used in the correct order
in the wrong order in the protocol. Repeat the purification procedure with a new sample.
g) New combination of reverse If enzymes are changed, it may be necessary to readjust the amount of carrier
transcriptase and Taq DNA RNA solution added to Buffer AVL.
polymerase used

DNA contamination
DNA and RNA present in To avoid co-purification of DNA, use of cell-free body fluids for preparation of
the sample viral RNA is recommended. Samples containing cells, such as cerebrospinal
fluid, bone marrow, urine and most swabs, should be made cell-free by
centrifugation or filtration. If using centrifugation, pellet the cells for 10 min at
1500 x g and use supernatant for isolation of viral RNA. If DNA-free RNA is
required, digest either the sample or the eluate with RNase-free DNase.
DNase in the eluate must be inactivated by heat treatment (15 min, 70°C).
General handling
a) Lysate not completely passed Using the spin protocol: Centrifuge for 1 min at full speed or until all the
through the membrane lysate has passed through the membrane.
Using the vacuum protocol: Insufficient vacuum was applied or the lid of the
spin column was closed during the vacuum step. Increase the vacuum, and
open the lid while applying the vacuum. If the vacuum pressure cannot be
increased, place the QIAamp Mini column in a clean 2 ml collection tube,
close the cap, and centrifuge at 6000 x g (8000 rpm) for 3 min or until the
lysate has completely passed through the membrane. Place the QIAamp Mini
column into another clean 2 ml collection tube, and discard the tube
containing the filtrate. Continue with step 7 of the spin protocol on page 27.
b) Clogged membrane Cryoprecipitates have formed in plasma due to repeated freezing and thawing.
Do not use plasma that has been frozen and thawed more than once.
c) Cross-contamination between To avoid cross-contamination when handling QIAamp Mini spin columns, follow
samples the guidelines in “Handling of QIAamp Mini columns” on page 20. Repeat the
purification procedure with new samples.
d) Vacuum pressure too Using a vacuum pressure that is too high may damage the QIAamp membrane.
high/low Using too low a vacuum pressure may cause reduced RNA yield and purity. Use
a vacuum regulator to adjust the pressure to –800 to –900 mbar for all vacuum
steps.

Appendix A: General Remarks on Handling
RNA
Handling RNA
Ribonucleases (RNases) are very stable and active enzymes that generally do not require
cofactors to function. Since RNases are difficult to inactivate and even minute amounts are
sufficient to destroy RNA, do not use any plasticware or glassware without first eliminating
possible RNase contamination. Great care should be taken to avoid inadvertently introducing
RNases into the RNA sample during or after the purification procedure. To create and maintain
an RNase-free environment, the following precautions must be taken during pretreatment and
use of disposable and nondisposable vessels and solutions while working with RNA.
General handling
Proper microbiological, aseptic technique should always be used when working with RNA.
Hands and dust particles may carry bacteria and molds and are the most common sources of
RNase contamination. Always wear latex or vinyl gloves while handling reagents and RNA
samples to prevent RNase contamination from the surface of the skin or from dusty laboratory
equipment. Change gloves frequently and keep tubes closed whenever possible. Keep purified
RNA on ice when aliquots are pipetted for downstream applications.
Disposable plasticware
The use of sterile, disposable polypropylene tubes is recommended throughout the procedure.
These tubes are generally RNase-free and do not require pretreatment to inactivate RNases.

Nondisposable plasticware
Nondisposable plasticware should be treated before use to ensure that it is RNase-free.

Plasticware should be thoroughly rinsed with 0.1 M NaOH, 1 mM EDTA* followed by RNase-
free water (see ”Solutions”, page 43). Alternatively, chloroform-resistant plasticware can be
rinsed with chloroform* to inactivate RNases.
Glassware
Glassware should be treated before use to ensure that it is RNase-free. Glassware used for
RNA work should be cleaned with a detergent,* thoroughly rinsed, and oven baked at 240°C
for at least 4 hours (overnight, if more convenient) before use. Autoclaving alone will not fully
inactivate many RNases. Alternatively, glassware can be treated with DEPC* (diethyl
pyrocarbonate). Fill glassware with 0.1% DEPC (0.1% in water), allow to stand overnight
(12 hours) at 37°C and then autoclave or heat to 100°C for 15 minutes to eliminate residual
DEPC.
Note: Corex® tubes should be rendered RNase-free by treatment with DEPC and not by baking.
This will reduce the failure rate of this type of tube during centrifugation.
Electrophoresis tanks
Electrophoresis tanks should be cleaned with detergent solution (e.g. 0.5% SDS),* rinsed with
water, dried with ethanol,*† and then filled with a solution of 3% H2O2.* After 10 minutes at
room temperature (15–25°C), the electrophoresis tanks should be rinsed thoroughly with
RNase-free water.
* When working with chemicals, always wear a suitable lab coat, disposable gloves and protective goggles. For more
information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
† Plastics used for some electrophoresis tanks are not resistant to ethanol. Take proper care and check the supplier’s
instructions.

Solutions
Solutions (water and other solutions) should be treated with 0.1% DEPC. DEPC is a strong, but
not absolute, inhibitor of RNases. It is commonly used at a concentration of 0.1% to inactivate
RNases on glass or plasticware or to create RNase-free solutions and water. DEPC inactivates
RNases by covalent modification. Add 0.1 ml DEPC to 100 ml of the solution to be treated
and shake vigorously to bring the DEPC into solution. Let the solution incubate for 12 hours at
37°C. Autoclave for 15 minutes to remove any trace of DEPC. DEPC will react with primary
amines and cannot be used directly to treat Tris* buffers. DEPC is highly unstable in the
presence of Tris buffers and decomposes rapidly into ethanol and CO2. When preparing Tris
buffers, treat water with DEPC first, and then dissolve Tris to make the appropriate buffer.
Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation.
Carbethoxylated RNA is translated with very low efficiency in cell-free systems. However, its
ability to form DNA:RNA or RNA:RNA hybrids is not seriously affected unless a large fraction
of the purine residues has been modified. Residual DEPC must always be eliminated from
solutions or vessels by autoclaving or heating to 100°C for 15 minutes.
Note: QIAamp Viral RNA buffers are not rendered RNase-free by DEPC treatment and are
therefore free of any DEPC contamination.
* When working with chemicals, always wear a suitable lab coat, disposable gloves and protective goggles. For more
information, consult the appropriate safety data sheets (SDSs), available from the product supplier.

Ordering Information
Product Contents Cat. no.
QIAamp Viral RNA Mini Kit For 50 minipreps: 50 QIAamp Mini Spin 52904
(50) Columns, Carrier RNA, Buffers and
Collection Tubes (2 ml)
QIAamp Viral RNA Mini Kit For 250 minipreps: 250 QIAamp Mini 52906
(250) Spin Columns, Carrier RNA, Buffers and
Collection Tubes (2 ml)
QIAcube Connect and QIAcube Connect accessories – for fully automated

sample preparation using QIAGEN spin-column kits
QIAcube Connect* Instrument, connectivity package, 1-year Inquire

warranty on parts and labor
Starter Pack, QIAcube Filter-tips, 200 µl (1024), 1000 µl filter-tips 990395

(1024), 30 ml reagent bottles (12), rotor
adapters (240), elution tubes (240), rotor
adapter holder
Filter-Tips, 1000 µl (1024) Sterile, Disposable Filter-Tips (racked) 990352
Rotor Adapters (10 x 24) For 240 preps: 240 Disposable Rotor 990394
Adapters; for use with QIAcube Connect
QIAamp Viral RNA Mini Additional Buffers and Reagents; for use 1048147
Accessory Set with at least 11 x QIAamp Viral RNA Mini
Kits (50; cat. no. 52904) or 5 x QIAamp
Viral RNA Mini Kits (250; cat. no. 52906)
on QIAcube Connect

Related products
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Vacuum Kit (50) Columns, QIAGEN Protease, Carrier
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Columns, QIAGEN Proteinase K, Carrier
RNA, Buffers, Extension Tubes (3 ml),
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QIAamp Media MDx Kit (12) For 12 x 96 preps: 12 QIAamp 96 Plates, 965752
Buffers, Proteinase K, S-Blocks, Disposable
Troughs, Racks with Elution Microtubes CL
(0.4 ml), Carrier RNA, Top Elute Fluid,
Caps, Tape Pad
QIAamp Virus BioRobot MDx For 12 x 96 preps: 12 QIAamp 96 Plates, 965652

Kit (12) RNase-free Buffers,§ QIAGEN Protease,
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EZ1® Virus Mini Kit v2.0 (48) For 48 preps: Reagent Cartridges (Virus Mini 955134
v2.0), Disposable Filter-Tips, Disposable Tip-
Holders, Sample Tubes (2 ml), Elution Tubes
(1.5 ml), Buffer AVE, Carrier RNA
EZ1 Virus Card v2.0 Preprogrammed card for EZ1 Virus v2.0 9017330
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MagAttract® Virus Mini M48 For 192 virus nucleic acid preps: 955336
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QIAamp RNA Blood Mini Kit For 50 RNA preps: 50 QIAamp Mini Spin 52304
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and Collection Tubes (2 ml)
Accessories
Buffer AW1 (concentrate, 242 ml Wash Buffer (1) Concentrate for 19081
242 ml) 1000 spin, 250 midi or 100 maxi preps
Buffer AW2 (concentrate, 324 ml Wash Buffer (2) Concentrate 19072

324 ml)
Buffer AVL (155 ml) For 250 preps: 155 ml Viral Lysis Buffer, 19073
5 x 310 µg Carrier RNA
Buffer AVE (108 x 2 ml) 108 x 2 ml Buffer AVE 1020953

Carrier RNA(poly A) Carrier RNA (poly A) 12 x 1350 µg, 1017647

12x1350µg-15-25°C,KG 15–25°C, KG
Collection Tubes (2 ml) 1000 Collection Tubes (2 ml) 19201

* All QIAcube Connect instruments are provided with a region-specific connectivity package, including tablet and
equipment necessary to connect to the local network. Further, QIAGEN offers comprehensive instrument service
products, including service agreements, installation, introductory training and preventive subscription. Contact
your local sales representative to learn about your options.
†
Fully automatable on QIAcube Connect. See www.qiagen.com/qiacubeprotocols for protocols.
‡
Other kit sizes are available; see www.qiagen.com.
§
Wash buffers are labeled with bar codes, and expiration date is stated on the Q-card in the kit.
For up-to-date licensing information and product-specific disclaimers, see the respective
QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are
available at www.qiagen.com or can be requested from QIAGEN Technical Services or your
local distributor.
Document Revision History

Date Changes
June 2012 Removal of risk and safety codes and statements.

General update into revised template.
December 2014 Changes to comply with GHS regulation, throughout the document.
March 2018 Update into Sample to Insight branding.

Change to step 3 of the spin protocol (page 28) and step 3 of the vacuum protocol (page 32),
to remove an outdated study reference.
January 2020 Updated text, ordering information and intended use for QIAcube Connect.

Limited License Agreement for QIAamp Viral RNA Mini Kit
Use of this product signifies the agreement of any purchaser or user of the product to the following terms:
1. The product may be used solely in accordance with the protocols provided with the product and this handbook and for use with components contained in the kit
only. QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included
within this kit except as described in the protocols provided with the product, this handbook, and additional protocols available at www.qiagen.com. Some of
these additional protocols have been provided by QIAGEN users for QIAGEN users. These protocols have not been thoroughly tested or optimized by
QIAGEN. QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third-parties.
2. Other than expressly stated licenses, QIAGEN makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties.
3. This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.
4. QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated.
5. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. QIAGEN
may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any
action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.
For updated license terms, see www.qiagen.com.
Trademarks: QIAGEN®, Sample to Insight®, QIAamp®, QIAcube®, BioRobot®, EZ1®, MagAttract®, MinElute®, UltraSens® (QIAGEN Group); Centricon®, UltraFree®
(Merck KGaA); Corex® (Corning, Inc.); Tween® (ICI Americas Inc.).
HB-0354-005 © 2020 QIAGEN, all rights reserved.

Notes

Notes

Notes

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HB-0354-005 QIAamp Viral RNA Mini Handbook 01/2020

Qiaamp Viral Rna Mini Handbook

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January 2020

QIAamp® Viral RNA

Intended Use .............................................................................................................. 5

Safety Information ....................................................................................................... 5

Cellular DNA contamination ............................................................................ 10

Warnings and precautions .............................................................................. 10

Sample volumes ............................................................................................. 11

Carrier RNA .................................................................................................. 12

Addition of internal controls ............................................................................. 12

Spin and vacuum procedures ........................................................................... 13

Automated purification of RNA on QIAcube Instruments ...................................... 13

Determination of yield ..................................................................................... 14

Equipment and Reagents to Be Supplied by User .......................................................... 15

Important Notes ........................................................................................................ 16

Preparation of reagents ................................................................................... 17

Handling of QIAamp Mini columns .................................................................. 20

Handling guidelines for the QIAvac 24 Plus ...................................................... 22

2 QIAamp Viral RNA Mini Handbook 01/2020

Protocol: Purification of Viral RNA (Spin Protocol) ......................................................... 27

Protocol: Purification of Viral RNA (Vacuum Protocol) .................................................... 31

Protocol: Sample Concentration .................................................................................. 36

Protocol: Purification of Cellular, Bacterial or Viral DNA from Urine ................................ 37

Troubleshooting Guide .............................................................................................. 38

Appendix A: General Remarks on Handling RNA......................................................... 41

Ordering Information ................................................................................................ 44

Document Revision History ......................................................................................... 47

QIAamp Viral RNA Mini Handbook 01/2020 3

Catalog no. 52904 52906

No. of preps 50 250

QIAamp Mini Spin Columns 50 250

Collection Tubes (2 ml) 150 750

Buffer AW1* (concentrate) 19 ml 98 ml

Carrier RNA (poly A) 310 µg 5 x 310 µg

4 QIAamp Viral RNA Mini Handbook 01/2020

CAUTION DO NOT add bleach or acidic solutions directly to the sample

QIAamp Viral RNA Mini Handbook 01/2020 5

6 QIAamp Viral RNA Mini Handbook 01/2020

QIAamp Viral RNA Mini Handbook 01/2020 7

Adsorption to the QIAamp membrane

8 QIAamp Viral RNA Mini Handbook 01/2020

Elution with Buffer AVE

Cellular DNA contamination

Warnings and precautions

10 QIAamp Viral RNA Mini Handbook 01/2020

QIAamp Viral RNA Mini Handbook 01/2020 11

Addition of internal controls

Refer to the manufacturer’s instructions to determine the optimal concentration. Using a

12 QIAamp Viral RNA Mini Handbook 01/2020

Automated purification of RNA on QIAcube Instruments

QIAamp Viral RNA Mini Handbook 01/2020 13

Determination of viral RNA length

14 QIAamp Viral RNA Mini Handbook 01/2020

For vacuum protocols

QIAamp Viral RNA Mini Handbook 01/2020 15

16 QIAamp Viral RNA Mini Handbook 01/2020

Addition of carrier RNA to Buffer AVL*

Where: n = number of samples to be processed simultaneously

y = calculated volume of Buffer AVL

z = volume of carrier RNA–Buffer AVE to add to Buffer AVL