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P4. Extraction of RNA

This document describes the process of extracting RNA from samples. There are two main methods - organic extraction using phenol/chloroform and column purification. The objectives are to isolate intact RNA for analysis. Samples can be immediately disrupted, frozen, or stored in RNAlater solution. The organic extraction method removes proteins, lipids and DNA using phenol/chloroform. Column purification binds RNA to a filter while washing away other components. Total RNA is then isolated using TRI reagent containing guanidinium thiocyanate and phenol, followed by precipitation with isopropanol and washing with ethanol before resuspension in water.

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Mehmood Iqbal
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58 views20 pages

P4. Extraction of RNA

This document describes the process of extracting RNA from samples. There are two main methods - organic extraction using phenol/chloroform and column purification. The objectives are to isolate intact RNA for analysis. Samples can be immediately disrupted, frozen, or stored in RNAlater solution. The organic extraction method removes proteins, lipids and DNA using phenol/chloroform. Column purification binds RNA to a filter while washing away other components. Total RNA is then isolated using TRI reagent containing guanidinium thiocyanate and phenol, followed by precipitation with isopropanol and washing with ethanol before resuspension in water.

Uploaded by

Mehmood Iqbal
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Extraction of RNA

(Practical # 4)

Dr. Zain Ul Abadeen


Assistant Professor
Ph.D. (Pathology), M.Phil., D.V.M.
RNA?
• RNA = Ribonucleic acid.
• A type of nucleic acid with only
one strand
- ribose instead of deoxyribose and
- using uracil instead of thymine (in
DNA).
• Provides the link between the
genetic information through protein
synthesis (serve as template for
protein synthesis).
Objectives

• Isolation of intact RNA is essential for many techniques used


in gene expression analysis such as:
➢Microarray analysis
➢Northern analysis
➢cDNA library construction
➢RT-PCR
Three ways to handle samples prior to RNA extraction

• Immediately disrupt the fresh samples


• Freeze the samples in liquid nitrogen
• Store the samples in RNAlater® solution
Total RNA extraction

• There are two methods for total RNA extraction


(1) Organic Extraction
(2) Column Purification
Organic Extraction
➢ Organic extraction (acidified phenol and
chloroform)
How it works?? ➢ Removes proteins, lipids, and DNA from the RNA
sample.
➢ RNA is then recovered by alcohol precipitation.
(i) Can be use for large or small sample sizes
Advantages (ii) Can modify extractions to remove high levels of fat
and protein from samples.

Disadvantage (i) Phenol and chloroform are hazardous


Column Purification
How it works??
• Glass filters bind the RNA while other cellular components
are washed away
• RNA is eluted in a highly purified form.
Column Purification

Advantages
(i) Rapid procedure
(ii) No organic solvents required
(iii) No alcohol precipitation needed.

Disadvantages
• Not as scalable as organic extraction methods.
RNA Extraction

Total RNA isolation reagent (TRI reagent solution) is Acid


Guanidinium Thiocyanate-Phenol-Chloroform.
RNA Extraction Steps
Cont…
Cont…
RNA Extraction Steps

The first step in RNA extraction is to break down the cells or


tissue so that the nucleic acids are released from the cells.

• Homogenization
Tissue: mortar and pestle
Cells: repetitive pipetting (resuspension)
Cont….

• RNA extraction methods use a powerful chaotropic salt


solution.
• This ‘lysis solution’ rapidly disrupts cells without destroying
their nucleic acids.
• Total RNA isolation reagent as ‘lysis solution’ combines
phenol and guanidine thiocyanate.
Phase Separation
• Homogenate (from homogenization step)
must be store for 5 min at room temp to
- permit the complete dissociation of
nucleoprotein complexes.
• Chloroform used to:
- Separate solution in aqueous phase,
interphase and organic phase
- RNA in aqueous phase, DNA (interphase)
and protein (organic phase)
Cont….

• Why use chloroform??


– Chloroform is organic solvent.
• So, lysed cell components that are hydrophobic will be
trapped in these solvent (e.g. membrane lipids, hydrophobic
polypeptide sequences (protein) or polysaccharides etc.)
RNA Precipitation
Isopropanol:
Precipitate RNA from the
aqueous phase.
• RNA precipitate (often visible
before centrifugation) forms a
gel-like or white-pellet on the
side and bottom of the tube.
RNA Wash & Solubilization

• 75% ethanol:
– Wash RNA pellet away from any salt residual.

• RNase free water:


– Clean RNA resuspended (RNase free water) to ensure
stability and long term storage.
Troubleshooting

If you get bad purity of RNA


➢Eliminating DNA contamination with DNase treatment

To remove residual genomic DNA and obtain highly pure RNA


➢Purify with column purification

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