Schwochow et al.

BMC Biotechnology 2012, 12:33

http://www.biomedcentral.com/1472-6750/12/33

METHODOLOGY ARTICLE Open Access

Efficient recovery of whole blood RNA - a

comparison of commercial RNA extraction
protocols for high-throughput applications in
wildlife species
Doreen Schwochow1,2*, Laurel EK Serieys1, Robert K Wayne1 and Olaf Thalmann1,3

Abstract
Background: Since the emergence of next generation sequencing platforms, unprecedented opportunities have
arisen in the study of natural vertebrate populations. In particular, insights into the genetic and epigenetic
mechanisms of adaptation can be revealed through study of the expression profiles of genes. However, as a pre-
requisite to expression profiling, care must be taken in RNA preparation as factors like DNA contamination, RNA
integrity or transcript abundance can affect downstream applications. Here, we evaluated five commonly used RNA
extraction methods using whole blood sampled under varying conditions from 20 wild carnivores.
Results: Despite the use of minute starting volumes, all methods produced quantifiable RNA extracts (1.4 –
18.4 μg) with varying integrity (RIN 4.6 - 7.7), the latter being significantly affected by the storage and extraction
method used. We observed a significant overall effect of the extraction method on DNA contamination. One
particular extraction method, the LeukoLOCK™ filter system, yielded high RNA integrity along with low DNA
contamination and efficient depletion of hemoglobin transcripts highly abundant in whole blood. In a proof of
concept sequencing experiment, we found globin RNA transcripts to occupy up to ¼ of all sequencing reads if
libraries were not depleted of hemoglobin prior to sequencing.
Conclusion: By carefully choosing the appropriate RNA extraction method, whole blood can become a valuable
source for high-throughput applications like expression arrays or transcriptome sequencing from natural
populations. Additionally, candidate genes showing signs of selection could subsequently be genotyped in large
population samples using whole blood as a source for RNA without harming individuals from rare or endangered
species.
Keywords: Whole blood, RNA preservation, RNA extraction, DNA contamination, Transcriptome sequencing, Globin
transcripts

Background in natural populations of vertebrates (i.e. [4,5]). Over the

Adapting to an ever-changing environment is one of the last several decades, molecular studies addressing signa-
most crucial functions of living organisms. Although we tures of selection have mainly focused on model organ-
have a profound understanding of how some adaptive isms or captive animals for which genome information
mechanisms have shaped phenotypic traits (i.e. [1-3]), became readily available and sample material can be eas-
their molecular foundation remains largely unexplored ily obtained [6-9]. Even though such studies provide fun-
damental knowledge for evolutionary biology, similar
* Correspondence: [email protected] studies on vertebrates in a natural context have thus far
1
Department of Ecology and Evolutionary Biology, University of California Los only marginally been explored. With the emergence of
Angeles, 2149 Terasaki Life Science Building, Los Angeles 90095, USA
2
Department of Animal Breeding and Genetics, Swedish University of
high-throughput applications such as expression arrays
Agricultural Sciences, BMC Husargatan 3, Uppsala SE75124, Sweden and next generation sequencing methods (NGS) new
Full list of author information is available at the end of the article

© 2012 Schwochow et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
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opportunities have arisen to investigate the genetic depletes the primary source of globin mRNA (reticulo-
mechanisms facilitating adaptation in natural popula- cytes). Blood samples cannot be frozen or stored in pres-
tions [10-13]. Moreover, the introduction of transcrip- ervation buffer for these approaches prior to processing
tome sequencing via RNAseq [14,15] has led to and hence these methods are impractical for field-based
important findings in the field of ecological genomics investigations of natural, free-ranging populations. How-
[16-18]. Owing to their high sensitivity, these novel tech- ever, a recently launched, commercially available whole
nologies require special precautions in sample handling blood filter system has been suggested to effectively filter
and preparation, a pre-requisite not often achievable for out reticulocytes while capturing and preserving leuko-
free-ranging, natural populations of vertebrates. For ex- cytes, the cell population most interesting for investiga-
ample, fresh tissue collection for RNA extraction can be tions of adaptive response to changing environmental
difficult for rare or endangered species. Although sam- conditions.
pling via fat or muscle tissue biopsies may be possible in As encouraging as these preservation methods appear,
some exceptional cases, the primary least invasive and to our knowledge, their performance has never been
abundant source of RNA in vertebrates remains whole evaluated by concurrent experiments on different spe-
blood. cies and using similar collection conditions. In the
Whole blood offers several advantages for transcrip- present study, we evaluated four commercially available
tome studies on non-model organisms but is surprisingly RNA preservation buffers applied to whole blood sam-
underrepresented in the recent literature. Blood has an ples in seven different carnivore species. Furthermore,
important role in mediating between the environment we explored the performance and the suitability of a
and the organism and hence exploring gene expression new whole blood filter system for application under field
profiles derived from blood may provide deeper insight conditions. All five sampling methods were evaluated
into immune response or stress metabolism [19-22]. with regard to RNA quality including DNA contamin-
Furthermore, compared to other tissues, blood is rela- ation and RNA yield as well as the integrity of the iso-
tively easy to obtain and small volumes can be collected lated RNA. In a proof-of-concept-experiment, we
with little harm to the animal. However, extracting use- modified and validated conditions of mRNA preparation
ful RNA from whole blood is hampered by several chal- from whole blood samples for NGS applications. Finally,
lenges. A first concern is the effective preservation of we provide guidelines on how to preserve and process
RNA. RNA is sensitive to endogenous and exogenous RNA extracts from whole blood sampled from wildlife
RNases, which rapidly degrade the nucleic acid resulting populations.
in changes in the expression profile and a potential loss
of rare transcripts immediately after sample collection Results
[23-26]. Solutions to this dilemma include immediate We evaluated different RNA extraction methods applied
snap freezing of the sample in liquid nitrogen or dry ice to whole blood sampled under field conditions that re-
[27,28], or storage in special whole blood preservation semble situations researchers face when investigating
buffers. Snap freezing presents an alternative for some non-model organisms in their natural habitat. We used
but may be difficult for more remote and unpredictable samples collected from 20 carnivores and RNA from
field situations. In contrast, storage buffers are easy to each individual was preserved using five methods (four
transport and are purported to increase the time before buffers and one filter each; except one individual for
freezing becomes necessary and thus provide researchers which we only collected blood for the four whole blood
with the flexibility to ship samples from the field. preservation buffers) resulting in a total of 99 RNA
A second challenge of using whole blood for transcrip- extractions (Additional file 1: Figure S1, Additional file
tome profiling is the heterogeneity of blood cells and the 1: Table S1).
high content of interfering hemoglobin mRNAs. Globin
mRNA in mammalian whole blood is estimated to ac- Presence of contaminants in the RNA extracts from blood
count for 70% of all mRNA transcripts (i.e. [29]), an ex- samples
cess that significantly affects the detection sensitivity of DNA might inadvertently be co-extracted with RNA and
less abundant mRNAs in high-throughput approaches thus negatively affect downstream applications. Since
and reduces the amount of target cDNA sequenced [29- most commonly used nucleic acid quantification proce-
32]. In order to minimize the confounding effect of glo- dures evaluate both RNA and DNA simultaneously, we
bin mRNA, methods have been suggested which reduce explicitly tested each RNA extract for potential DNA
the abundance of those transcripts prior to high- contamination by means of PCR amplifications.
throughput sequencing. These methods range from bio- In total, we examined four PCRs for each extract and
tinylated globin capture oligos, to a laborious fractionat- found 62 of 80 whole blood non-filtered extracts to con-
ing of whole blood using microcentrifuges which tain traces of DNA contamination. When considering
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bands in the expected sizes, 44.0 – 74.0% of the total Bioanalyzer. This procedure effectively removed all detect-
PCRs resulted in amplification products (Table 1). The able traces of DNA contaminants.
RiboPure™ kit samples showed the highest percentage of Besides DNA contamination, the presence of add-
amplification and hence the largest amount of DNA itional contaminants such as proteins, salt and organic
contamination. The successive PCRs were distributed compounds were determined by measuring the absorp-
among 17 of the 20 tested extracts, leaving only 15.0% tion at wavelengths A260/280 and A260/230 using
free of DNA contamination. Of those RiboPure™ extracts Nanodrop. We observed significant variation between
contaminated with DNA, a surprising 30.0% gave pro- the different procedures (ANOVA: p = 3.678e-09 for
ducts for all four test PCRs. A modest amount of DNA A260/280 and p = 6.103e-08 for A260/230). The A260/
contamination was observed for samples extracted with 280 ratio averaged between 1.9 and 2.1 for all extraction
the PAXgene™ blood kit (overall 54.4% positive PCRs), kits (Table 1), while more variation was observed for
and lastly, TRIzolW LS and RNeasyW methods gave only A260/230 ratios. Only the RiboPure™ kit resulted in a
43.8% and 40.0% positive PCRs, respectively. A260/230 ratio of 2.1 indicating samples free of organic
We additionally investigated the performance of a compounds, followed by TRIzolW LS (1.3 ± 0.5), PAX-
commercially available kit (LeukoLOCK™) that filters gene™ (1.2 ± 0.6), RNeasyW (0.9 ± 0.6) and LeukoLOCK™
leukocytes and depletes the amount of reticulocytes with the lowest ratio (0.8± 0.5).
prior to extraction. We found that among the 19 pre-
filtered extracts only four samples yielded PCR amplifi- RNA yield and integrity
cations. This corresponds to only 11% amplification rate After complete depletion of contaminant DNA, we
(Table 1) and is the lowest contamination rate observed determined the amount of extracted RNA. Starting from
in this study. Despite this low degree of contamination, minute volumes of only 500 μl whole blood (Additional
two positive samples amplified three out of four test file 1: Figure S1), we obtained the following RNA yields
products, a result indicating substantial contamination with significant variation between the applied methods
within those affected extracts. (ANOVA: p = 4.653e-10): 5.0-43.9 μg using RiboPure™;
When testing if the extraction method, the species or 0–6.2 μg for the RNeasyW; 0.1-10.2 μg for the PAXgene™
individual factors of each animal affected the DNA con- and 0.2-15.1 μg for samples extracted using TRIzolW LS
tamination in the 99 RNA samples, only the extraction (Table 1, Figure 2A). The RNA yield range adjusted for
method revealed a significant correlation (ANOVA: 500 μl filtered blood using LeukoLOCK™was amongst the
p = 2.948e-07; Pearson’s Chi-squared test: p = 1.254e-09). lowest with 0.1- 3.7 μg (Table 1, Figure 2).
We also detected severe DNA contamination through We further investigated the integrity of each RNA
visual inspection of electropherograms in eight out of the extract as indicated by the RIN factor and observed the
99 extracts processed on Agilent’s Bioanalyzer (Figure 1). following average values: 4.6 ± 2.3 for samples extracted
Interestingly, among the eight samples, the majority were using RiboPure™ (stored in RNAlaterW); 6.9 ± 2.6 RNeasyW
extracted using the RiboPure™ method and underscore the (stored in RNAprotectW); 7.7 ± 1.2 for PAXgene™ (stored
tendency of this method to co-extract DNA. Finally, in in PAXgene™ blood buffer), 6.2 ± 2.9 TRIzolW LS (stored in
order to further reduce the DNA contamination of the TRIzolW LS; Table 1, Figure 2B) and lastly a RIN value of
RNA extracts, we performed a second, more rigorous 7.6 ± 1.9 for the filtered blood (stored in RNAlaterW). The
DNase treatment of all extracts that yielded more than values for RNA integrity differed significantly between
two successful PCR amplifications as well as the eight methods (ANOVA: p = 0.007). In comparison to RNeasyW
RNA samples showing visual DNA contamination on the and PAXgene™, whole blood samples extracted with
Table 1 RNA integrity, yields and DNA contamination for each tested preservation buffer and extraction method
Approach Preservation Extraction RIN RNA 260/ 260/ DNA contamination
buffer kit/protocol yield 280 230
% Amplification1 % Individuals
range
with min.
(μg/
1 amplicon2
500 μl
blood)
Whole blood RNAlaterW RiboPure™ 4.6± 2.3 5.0- 43.9 1.9± 0.1 2.1± 0.2 74.0 85.0
W W
RNAprotect RNeasy 6.9± 2.6 0.0- 6.2 2.1± 0.2 0.9± 0.6 40.0 80.0
PAXgene™ PAXgene™ 7.7± 1.2 0.1- 10.2 2.1± 0.2 1.2± 0.6 54.4 95.0
W W
TRIzol LS TRIzol LS 6.2± 2.9 0.2- 15.1 1.9± 0.1 1.3± 0.5 43.8 80.0
Filtered blood RNAlaterW LeukoLOCK™ 7.6± 1.9 0.1- 3.7 2.0± 0.1 0.8± 0.5 11.0 20.0
1
Percentage of PCRs that resulted in positive amplification. Each PCR performed in four reactions that amplified were counted.
2
Percentage of individuals in which a minimum of a single amplification occurred.
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Figure 1 Electropherograms exemplifying quality differences in various RNA extracts. Agilent 2100 Bioanalyzer electropherogram obtained
from one individual extracted using the Mouse RiboPure™ kit without DNase treatment (A); after one DNase treatments (B); and using the
RNeasyW kit including one DNase treatment (C). The broadened peak for the 28 s ribosomal RNA in (A) indicates substantial DNA contamination
of the RNA samples. After DNase treatment, traces of partially sheared DNA are still visible below the 18 s peak (B). Samples extracted using the
RNeasyW kit including one DNase treatment, give a clean electropherogram with well-defined 18 s and 28 s peaks with a high RIN value (C).

TRIzolW LS or RiboPure™ showed a broad range of well- field (5.8 ± 1.2). Furthermore, we observed a significant
preserved RNA samples to RNA samples totally degraded. effect of the sample collector as well as the species
Due to low RNA concentrations in 12 samples, we lowered (p = 0.002 and p = 5.872e-06, respectively) but these two
the threshold parameters in the Agilent Bioanalyzer soft- factors are not mutually exclusive (Additional file 1:
ware for these particular samples in order to obtain a RIN Table S1).
value. These samples included a total of seven study ani-
mals (three bobcats, two foxes, one sea lion and one coy- Hemoglobin depletion
ote) and all extraction/ storage methods were affected. In order to evaluate a potential reduction of hemoglobin
We found a trend towards higher average RIN values transcripts in the filtered extracts, we used a quantitative
in extracts collected in a lab-like facility (i.e. in zoo facil- PCR assay to estimate the relative ratio between
ities; 7.4 ± 1.4) when compared to those collected in the hemoglobin and three housekeeping genes and
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Figure 2 RNA yield and RIN values. RNA yield and RIN values for the five different RNA extraction protocols. (A) RNA yield (in μg) obtained
after one or (if required) two DNase treatments from 500 μl blood using four different protocols/ kits for RNA extraction from whole blood as
well as the LeukoLOCK™ filter system. (B) RIN values of the RNA extracted from blood samples preserved in different buffers. Please note that
samples collected with the RNA filter “Leuko” were stored in RNAlaterW just as the whole blood samples “RNAlater”. The RIN values of both
collection methods differ dramatically and represent both the upper and lower limit of the performance of all tested preservation buffers.

compared them to the values derived from the four percentage of singletons (63.71%) was found in sample
other whole blood extraction methods. The average log A1, which in turn showed the second largest number of
transformed ratio for hemoglobin in filtered blood was total reads (Table 2). We detected 36.47% and 28.22%
significantly higher than that of whole blood (Figure 3, singletons in samples A2 and B1, respectively. This re-
Student’s t-test, p = 0.005). On average, hemoglobin was sult suggests that the number of total reads does not ne-
reduced by 70% in the extracts processed through the filter. cessarily determine the abundance of singletons, but
When three outliers are excluded, the average reduction additional factors such as tissue type or stochastic effects
increased to 84%. Interestingly, we found a strong correl- need to be considered.
ation between the log transformed ratios and the species
used in this study (ANOVA: p = 4.393e-16). However, the
species were sampled by different researchers and this
could consequently also contribute this association.

454 sequencing results

Four cDNA samples were prepared differently for use
on a 454 GS FLX Titanium sequencer (Table 2). We first
assembled all raw reads per sample. Sample B2 showed
the lowest number of reads (17,047) with 4,391 reads
(25.76%) assembled into 309 contigs. Sample A2 had the
largest number of reads (38,601). Fifty percent of those
reads (19,318) were assembled into 2,286 contigs. The
remaining two samples A1 and B1, yielded 30,145 and
21,219 reads, from which 30.31% and 61.62% built 1,066
and 275 contigs, respectively. Interestingly, the number
of singletons (reads only occurring once and not
assembled in any contig), did not correlate with the total Figure 3 Log transformed ratio of hemoglobin transcripts. Log
number of reads per sample. For example, the sample transformed ratio of hemoglobin transcripts abundant in whole
blood extracts as well as those derived from the LeukoLOCK™ filter
with the lowest number of reads (B2) yielded the highest
system.
number of singletons (65.51%), the second highest
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Table 2 Sample preparation procedure and 454 sequencing results

Sample ID Preparation steps # raw reads # reads in contigs # contigs # singletons % globin
reads
A1 ribo-minus > 1st & 2nd strand cDNA 30,145 9,138 1,066 19,205 5.46
synthesis > NORM > 2nd amp
A2 wholeRNA > 1st&2nd strand cDNA 38,601 19,318 2,286 14,079 1.95
synthesis > 2nd amp > NORM > 3rd amp
B1 globin-minus > ribo-minus > 1st&2nd 21,219 13,075 275 5,987 24.10
strand cDNA synthesis
B2 globin-minus > ribo-minus > 1st&2nd 17,047 4,391 309 11,168 0.35
strand cDNAsynthesis > NORM > 2nd amp
# counted.
Abbreviations used for preparation steps are explained in the Supplementary Information Additional files.

We investigated the effectiveness of the different DNA contamination is particularly worrisome if ex-
whole blood sample preparation methods to exclude pression profiles are generated with high-throughput
hemoglobin transcripts. We extracted the genomic methods because even a single contaminating molecule
regions harboring hemoglobin genes from the publicly can be detected, making sequencing efforts less efficient
available dog genome [33] and mapped all raw reads of and potentially leading to false biological conclusions.
each sample to this hemoglobin reference. Although we Our DNA contamination tests did reveal a substantial
used various methods to deplete globin transcripts from amount of DNA co-extracted with all RNA extraction
each sample, sequences mapping to the reference region protocols for whole blood. Between 80.0-95.0% of the
could be found in every sample (Table 2). A surprising whole blood RNA extracts resulted in at least one single
24.06% of all reads obtained from sample B1 mapped to DNA amplicon (Table 1). We observed significant differ-
the reference region, followed by 5.46% of the reads in ences in the extent of contamination depending on the
sample A1 and 1.95% of sample A2. The least number of extraction method used, which might reflect the efficacy
reads mapping to the globin reference was found in of the different techniques to remove DNA. For
sample B2 (0.35%), suggesting that the preparation instance, the RiboPure™ protocol is based on a guanidi-
method applied to this sample most effectively depleted nium thiocyante- phenol- chloroform homogenate that
globin transcripts. is extracted under low pH. This procedure is known to
Lastly, we were interested in the number and identity avoid co-extraction of DNA [34] and therefore the
of genes that are present in each of the four samples. observed 85.0% contaminated extracts were quite
The two samples with the largest numbers of contigs, unexpected. Such heavy contamination might be the
A2 and A1 also yielded the largest numbers of assigned result of carryover DNA from the interface during the
genes, 486 and 292 respectively. However, a higher phenol-chloroform extraction used in this procedure.
percentage of assigned contigs were observed in samples The applied DNase depletion protocols recommended
B1 with 57.0% (157 out of 275 contigs) and B2 with for each of the tested method should in theory have
35.0% (109 out of 309 contigs). We extracted the removed moderate amounts of DNA (<50 μg DNA/ ml
number of reads per gene in order to evaluate a poten- RNA extract). Surprisingly, irrespective of the extraction
tial over-representation of particular genes in the method, an additional DNase treatment was required in
sample. Additional file 1: Table S2 summarizes the top order to ultimately deplete any trace of DNA, which
10 genes with the largest number of reads per sample indicates severely contaminated RNA extracts. This is
and in concordance with the results described above, somewhat alarming and in agreement with results
globin genes can be found in three samples, indicating reported for other RNA extraction methods [35]. Our
its omnipresence. finding implies that great care should be taken in order
to generate endogenous RNA sequence reads on any of
Discussion the NGS platforms.
Evaluation of RNA preservation buffers and extraction Aside from DNA, other contaminants of RNA extracts
kits for whole blood derive from incomplete removal of cellular components
The principle aim of this study was to assess the such as proteins, lipids and carbohydrates or traces of
performance of commercially available kits and proto- salt and organic solvents stemming from the extraction
cols commonly used to preserve and extract RNA from procedure itself. With regard to protein depletion in
blood samples. We used total RNA yield, RNA preserva- RNA extracts, determined by the A260/280 ratio, all
tion (RIN) and the degree of DNA contamination as methods yielded samples with a ratio averaging above
measures of performance. 1.9, which is considered suitable for NGS [36,37]. In
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contrast, when considering other organic substances (i.e. kits (samples stored in RNAprotectW) both yielded
Phenol) and aromatic compounds (i.e. Trizol) that ab- extracts with the highest RIN values, which makes them
sorb at 230 nm wavelength, only the RiboPure™ kit most suitable for sensitive downstream applications.
yielded satisfying results with a A260/230 ratio above These two methods also produced the extracts with the
1.8, a threshold indicating a low level of contamination. narrowest range of RNA preservation variation and are
Although some studies have reported reduced efficiency among the easiest to use, which highlights the suitability
of sensitive, downstream applications due to such con- of these methods whenever samples are collected under
taminants [36] subsequent sample processing might difficult, non-laboratory conditions.
eliminate these contaminants and the actual effect could Notably, the two methods resulting in the highest yield
be negligible when compared to that caused by DNA of RNA (TRIzolW LS, and RiboPure™ with samples
contamination. stored in RNAlaterW) produced the extracts with the
After depletion of interfering DNA, an accurate lowest and most widely scattered RIN values rendering
quantification of extracted RNA was possible. Depend- its suitability for high-throughput applications question-
ing on the extraction kit, the RNA yield for 500 μl blood able. This was surprising since both storage buffers have
is supposed to range between 1.6 μg up to 55 μg. While extensively been used to preserve RNA from a broad
the yield obtained for PAXgene™ samples is similar to range of animal tissues including brain, liver, testis,
that suggested by the manufacturers (0.1-10.2 μg/500 μl tissue culture cells or white blood cells in many different
blood vs. 1.6- 3.2 μg/500 μl blood as tested by the manu- species (e.g. [9,39-41]). The TRIzolW LS reagent used
facturer), the amount of RNA retrieved using the RNea- herein is a ready-to-use solution for isolation of total
syW kit was much lower than stated (0–6.2 μg/500 μl RNA from liquid samples of different origins (TRIzolW
blood vs. 25–35 μg/ 500 μl blood). Many factors, such as LS). One possible explanation for the weak performance
the individual hydration condition of an animal or blood of this method might be the unconventional application
density differences in certain parts of the body [34] can of the storage buffer under field conditions. More
influence the amount of RNA obtained from blood sam- precisely, the solution was aliquoted into RNase free
ples and help to explain the observed variation. tubes and provided to collectors. The time elapsed until
Intriguingly, our study showed that samples extracted actual usage of the buffer for sample collection ranged
using organic solvents like phenol/chloroform (e.g. Ribo- from a few weeks to two months and we observed a
Pure™ kit) or TRIzolW LS reagent resulted in the highest decrease in volume in some of these tubes, probably due
RNA yields (see also [36,38], Figure 2A). While the com- to evaporation. Furthermore, it is recommended to keep
bination of phenol/chloroform and RNA bound to glass the TRIzolW LS solution away from higher ambient
fiber columns (as employed by the RiboPure™ kit) temperatures and light, factors often unmanageable in
seemed to be very efficient in retrieving RNA [36], the field sites and shown to affect DNA recovery from fecal
purely column-based approaches not incorporating samples [42]. In fact, we found a significant difference
organic solvents (PAXgene™ and RNeasyW) yielded about between the average RIN values of TRIzolW LS samples
five times lower average RNA quantities. collected under field conditions (RIN = 4.4) and in zoo-
We observed a significant effect of the collector on the like facilities (RIN = 7.9; p = 0.005). The inefficiency of
RNA yield (ANOVA: p = 0.02) as well as the preservation TRIzolW to serve as preservation agent was previously
of the RNA (ANOVA: p = 0.002). The reasons for this also reported by Chiari and Galtier [34]. In summary,
pattern might include individual differences in sample apart from its toxicity, our findings suggest that TRIzolW
handling procedures. However, we need to stress the fact LS would not be the method of choice for blood
that the effect of collectors and species could not be dif- collection in the field, especially in warmer climates.
ferentiated since in most cases samples from one species Another commonly used RNA preservation solvent for
were collected by a single person under unique condi- whole blood samples is RNAlaterW (e.g. [9]). We found a
tions (Additional file 1: Table S1). Consequently, our surprisingly weak performance of this preservation
results imply that either single or correlated effects may method. According to the manufacture’s information
potentially cause differences in RNA yield. high amount of proteins present in whole blood samples
Aside from retrieving sufficient RNA quantities, its in- might lead to insoluble precipitation with RNAlaterW.
tegrity is yet another, perhaps even more important One observation supporting this possibility was the high
factor affecting downstream applications [37]. If not amount of coagulated blood sampled in RNAlaterW, a
stored properly, low abundance and long transcripts are pattern not prevalent in any of the other preservation
rapidly degraded by tissue specific RNases. This process buffers storing the same blood volume from the same
can affect RNA integrity and result in changes of the animals. Since the preservation process of RNAlaterW
biological expression profile [23-26]. The PAXgene™ relies upon the permeability of the tissue cells, it is
(samples stored in PAXgene™ blood buffer) and RNeasyW possible that large blood precipitates hamper the
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chemical’s ability to pass through the cell membrane and blood and warrants special treatment [29,32,44]. A sys-
interact with the RNA. This is in accordance with other tem such as LeukoLOCK™ results in the removal of 70%
studies reporting that the size of solid tissue material of hemoglobin transcripts. However, other extraction
stored in RNAlaterW negatively affects the RNA integrity methods require additional treatments to deplete these
[36,37]. However, whenever RNAlaterW was used in con- transcripts which may be necessary to allow the detec-
junction with the filter system it served as the best pre- tion of potential candidates of selection such as low
servative for RNA as we discuss below. abundant transcripts of disease related genes [29,45].
Methods for hemoglobin reduction are readily available
General performance of a filtering method and its effect but in most cases are designed exclusively for model
on hemoglobin reduction organisms.
We tested the performance of another RNA extraction
method that a priori filters leukocytes from whole blood Efficiency of RNA preparation methods for NGS
and by depleting an excess of hemoglobin mRNA, it ul- Whether finding the causative mutations or simply cata-
timately reduces the amount of downstream inhibitors loguing the wealth of expressed genes is a priority, nor-
in RNA extracts. However, this filtering procedure malizing the RNA extract is beneficial [46,47]. In
comes with a cost as the average yield of 1.2 μg/ 500 μl agreement with previous studies, we found that normal-
blood in the LeukoLOCK™ filter system was the lowest ized samples produced the highest number of contigs
in our analysis. Although low in quantity, the RNA itself but also singleton reads, which are equivalent to rare
was well preserved as indicated by high RIN values transcripts that might harbor valuable information
(Table 1, Figure 2B). Only four out of 19 samples otherwise not accessible [16,48]. Normalization of RNA
showed a RIN below 7, and we argue that these rare extracts effectively reduces highly abundant transcripts
cases might be due to inappropriate handling of the filter from the blood but in cases such as the omnipresent
system by collectors in the field. The handling process hemoglobin, additional measures need to be considered.
itself is more elaborate and requires a certain degree of Various methods depleting hemoglobin prior to sequen-
laboratory skill and precautions. It is a multi-step pro- cing exist and can be readily applied at different stages
cedure, which is cumbersome in the field, particularly if of sample preparation. We show that the application of
researchers aim to preserve the RNA immediately after such methods in combination with normalization con-
blood collection. In field settings that require animal tributed to a substantial reduction of reads representing
handling, researchers are often more focused on the ani- hemoglobin when compared to the 25.0% hemoglobin
mal itself and are shorthanded so the immediate proces- reads apparent in the non-normalized sample (B1). This
sing of blood through the filter post collection may be effect of normalization is only beneficial in qualitative
difficult. assessments of an organism’s transcriptome but should
An encouraging aspect of this particular system was be avoided whenever quantification of gene expression is
the low amount of DNA contamination observed in all desired.
RNA extracts. Only four of 19 samples yielded positive
test PCRs but only two of them amplified in more than Conclusions
three PCRs. The actual co-extraction of DNA from fil- The ultimate decision about which RNA extraction method
tered blood might be lower due to the efficient removal to apply may reflect a balance between performance, the
of the majority of erythrocytes. Whereas, mature mam- anticipated laboratory effort, the project’s logistics and
malian erythrocytes contain neither a nucleus nor cell finances and the scope of the project. Considering all tests
organelles like mitochondria, immature reticulocytes employed in this study, we conclude that the most
released from the bone marrow still harbor a nucleus. promising RNA storage and extraction method is the
These cells constitute about 1% of the total number of LeukoLOCK™ filter system. Besides its performance, the
circulating erythrocytes, which themselves number be- method includes practical amenities well suited for field
tween four and six million in a single μl of human blood. applications. First, the RNAlaterW soaked membrane of the
This leaves about 40,000 – 60,000 nuclei exclusively filter system allows flexibility in handling time as the RNA
derived from immature red blood cells and given the is stable for 24 hours at 37 °C, one week at 25 °C and one
sensitivity of PCRs (i.e. [43]), this number is sufficient to month or more at 4 °C. Second, the LeukoLOCK™ system
result in positive test PCRs whenever these cells are not works reliably even with small amounts of starting material.
depleted a priori. The system was originally designed for 9 ml human blood
Insufficient elimination of reticulocytes from whole but we have demonstrated that much smaller volumes
blood RNA extracts not only affects DNA contamination (1 ml whole blood) are sufficient as well and might
but might also change the ratio of highly abundant ver- therefore become the method of choice for small species or
sus rare transcripts. Hemoglobin is highly expressed in individuals that are difficult to handle or suffering from
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stress. One caveat about the system is the complex sample Each individual’s blood was sampled at the same time for
processing, which requires a certain familiarity with the all five approaches simultaneously, except for one coy-
steps in the protocol. However with appropriate practice ote where we failed to obtain a LeukoLOCK™ sample
and precautions the benefits of this system outweigh the (see Additional file 1: Table S1). The samples were col-
extra effort, particularly in remote areas with limited access lected according to national guidelines for handling
to electricity and refrigeration. animals and each institution has acquired the respect-
The second best performing method was the PAXgene™ ive permits required for sampling wild animals. All
kit. The preservation buffer is intended for stabilization of procedures were performed by highly qualified
blood for as much as three days at 18- 25 °C or for 5 days personnel and followed strict ethical guidelines.
at 2-8 °C. This method has been designed for medical
applications and is therefore more expensive than other Laboratory methods
kits. We would like to stress that great care has to be RNA extraction and quality assessment
taken to ensure a sufficient removal of DNA co-extracted The samples were extracted in three batches using com-
with this RNA extraction kit. mercially available kits by following manufacturer’s pro-
We show that whole blood can be a valuable resource tocols (Table 1). However, some modifications were
for transcriptome studies and that the efficiency of NGS incorporated when using the PAXgene™ buffer as sug-
technologies can be maximized by employing appropri- gested for low amounts of starting materials [49]. Spe-
ate library preparation methods (i.e. normalization). cies and order of samples were randomized between
Consequently, whole blood is a promising and easy batches as well as during extractions.
accessible RNA source that has great potential for NGS Extracted RNA samples were subjected to kit specific
studies in ecological and conservation genetics. or recommended DNase treatments. The efficiency of
DNA removal was determined in “no-RT” control PCR
Methods using a regular polymerase. We targeted four loci (see
Sample collection for RNA preservation and extraction section below) by using the following conditions. A mas-
evaluation termix was prepared with 0.2 U TaqGold (Applied
Whole blood samples were collected from seven carnivore Biosystems), 1x buffer Gold, 1.5 mM MgCl2, 4% DMSO,
species, including four Mexican wolves (Canis lupus 0.2 mM each dNTP, 0.5 μM forward and reverse primer
baileyi), four grey foxes (Urocyon cinereoargenteus), three mix and 1 μl of the non-transcribed RNA. The PCR was
bobcats (Lynx rufus), two coyotes (Canis latrans), four performed on an Eppendorf Mastercycler EP Thermal
California sea lions (Zalophus californianus), two harbor cycler (Eppendorf) using the following cycling steps: initial
seals (Phoca vitulina) and one California sea otter (Enhydra denaturation at 94 °C for 9 minutes, denaturation at 94 °C
lutris) in different facilities/ field settings (Additional file 1: for 20 seconds, annealing temperature 56 °C for 20 sec-
Table S1). In order to stabilize and preserve the RNA, onds, extension at 72 °C for 20 seconds; final extension at
we first collected 3 ml whole blood from each individ- 72 °C for 4 minutes; 40 cycles total. PCR amplification
ual into an EDTA coated blood tube. Using a sterile was evaluated on a 2% Agarose gel stained with SYBR Safe
syringe, approximately 0.5 ml of whole blood was trans- DNA gel stain (Invitrogen) and visualized under UV light.
ferred into each of four tubes containing unique RNA Samples that successfully amplified in at least two out of
preservation buffers. These buffers were: RNAlaterW the four PCRs were subjected to an additional DNase
(Ambion), RNAprotectW (Qiagen), PAXgene™ blood buffer treatment using 4 U of rDNaseI (Ambion) according to
(PreAnalytiX, Qiagen) and TRIzolW LS Reagent (Invitrogen) the manufacturer’s recommendation.
(Additional file 1: Figure S1). Whereas RNAlaterW and In order to evaluate the RNA yield, 10 μl of 1:10 diluted,
RNAprotectW buffers and tubes were used as provided by DNA-free RNA was used in a Quant-iT™RiboGreenW
the manufacturer, 1500 μl of TRIzolW LS Reagent was RNA reagent assay (Invitrogen), which uses a dye that
aliquoted into RNase free 2 ml tubes assuring a blood to intercalates with nucleic acids and absorbance (260 nm)
buffer volume ratio of 1:3. A similar volume adjustment could be measured at with a multimode DTX880 plate
was performed according to Carrol et al. (2007) using the reader (Beckman Coulter). RNA integrity was assessed at
PAXgene™ blood buffer [49]. The remaining 1 ml whole the UCLA Sequencing and Genotyping Core using the
blood was processed directly from the EDTA tube through RIN values calculated on an Agilent 2100 Bioanalyzer
a pre-assembled LeukoLOCK™ filter system (Ambion). The (Agilent Technologies).
filter was flushed with 2 ml PBS (Ambion) and subse-
quently with 2 ml RNAlaterW. Once the filter was saturated cDNA synthesis and target gene amplification
with RNAlaterW, it was sealed until further use. All samples In order to assess whether prior filtering (LeukoLOCK™
were shipped to the lab at UCLA immediately after collec- filter system) would efficiently reduce the amount of
tion in the field and kept at −20 °C until further processing. hemoglobin transcripts compared to the whole blood
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approaches, we performed a qPCR targeting ß- Data analyses

hemoglobin as well as three housekeeping genes for all DNA contamination, RNA yield and integrity, and
extracted samples. A volume of 4 μl undiluted DNA-free hemoglobin reduction
RNA was reverse transcribed using SuperScript III First- To first evaluate the extent of DNA contamination of
Strand Synthesis Super Mix (Invitrogen). Since random each RNA extract, we used two different analytical
hexamers have shown to result in an overestimation of approaches. Assuming that higher concentration of
mRNA copy number by up to 19-fold [50], we used DNA in an extract is more likely to yield amplification
oligo(dT) primers instead. success, we first counted the number of positive PCR’s
Primers were designed based on a multi species for each extraction method and performed a Pearson’s
alignment and are available upon request. Previous Chi square test. We further calculated the ratio of
findings in humans [51] suggest that three expressed positive PCRs for each sample and performed an
housekeeping genes are sufficient to normalize qPCR ANOVA using the statistical software package R
data from leukocytes. Here we used Ubiquitin C (Ubc), (http://www.R-project.org).
Phospholipase A2 (YWHAZ) and the human acidic Total RNA yield was estimated using the Quant-
ribosomal protein (HuPo; [52]). iT™RiboGreenW RNA reagent assay comparing the esti-
1st strand cDNAs were diluted 1:100. Additionally, mates of RNA concentrations against a provided stand-
samples that were filtered through the LeukoLOCK™ sys- ard and adjusted for a starting volume of 500 μl whole
tem were first diluted 1:2 in order to account for the blood. To obtain a reliable and comparable measure for
1 ml whole blood starting material. 1 μl of each dilution the integrity of all extracted RNA samples, we used the
was used in a 10 μl reaction of LightCycler 480 SYBR RNA integrity number (RIN), a measurement that uses
Green I Master mix (Roche). All samples from one indi- the entire electrophoretic trace of each RNA sample. We
vidual were run at the same time in duplicates and on used the Agilent 2100 Bioanalyzer and accompanied
the same plate to reduce nuisance parameters such as software which calculates the RIN values within a range
technical variations within individuals. We included two of 0 and 10, where 0 represents a totally degraded and
DNA samples as a positive control/ positive calibrator 10 a completely preserved RNA sample [53]. An
on each plate. All primers were used in a 0.5 μM end ANOVA was performed in order to find correlations be-
concentration. The LightCycler protocol included 5 min- tween the extraction methods and RNA yield, the stor-
utes activation at 95 °C, 10 seconds denaturing at 95 °C, age buffer and RIN factor as well as other variables such
primer annealing for 15 seconds at 55 °C, elongation for as collection conditions.
30 seconds at 72 °C and a melting curve (95 °C for To assess the amount of hemoglobin transcripts for
5 sec, 65 °C for 2 min and 97 °C). The last step was a the whole blood and the filtered approach, we processed
cooling step at 40 °C for 10 sec. The samples were sub- the outputs from the LightCycler runs (using LightCycler
jected to 50 cycles. In addition to the qPCR set up, we 480 software) by first undertaking a melting curve analysis
evaluated the PCR efficiency for each primer pair by in order to determine that each PCR worked properly and
including 12 diluted DNA samples (original concentra- no unspecific amplification occurred [54]. Samples that
tion, 1:2, 1:10 and 1:100). didn’t result in clean melting curve patterns (i.e. double
peaks indicating nonspecific amplification) were
Sample preparation for 454 sequencing omitted from further analysis and the qPCR was
Blood samples from two confined wolves (Mexican and repeated. In the next step, we estimated the relative
Arctic wolf ) were collected into PAXgene™ blood tubes ratios between our target (ß- hemoglobin) and the refer-
and prepared for sequencing utilizing Roches’ 454 GS ence genes (HuPo, Ubc, YWHAZ) using the relative
FLX Titanium platform following four different proce- quantification software (Efficiency-Method, Applied
dures. After the extraction of RNA, each extract was Bioscience). This approach compensates for differences
treated differently (Table 2), allowing us to compare the in target and reference gene amplification efficiency
performance of different methods such as globin reduc- within an experiment and between experiments. The
tion or enzymatic normalization. The actual 454 GS FLX PCR efficiency for each primer pair was evaluated once
Titanium library preparation steps included blunt ending and used as an external standard throughout the
of small DNA fragments, 454 GS FLX Titanium adapter experiment. The target was paired with all three refer-
ligation, various purification steps, emulsion PCR and a ence genes and the geometric mean of the resulting
final release of single-stranded DNA fragments. A more ratios was calculated. By equalizing the average ratios
detailed description is provided in the Supplementary of the whole blood approaches with 100% of globin
Information. As a proof of concept, we sequenced each transcripts, we were able to determine the average
prepared library on a single 1/16 lane of a 454 GS FLX reduction of ß- hemoglobin in the filtered samples. To
Titanium run. evaluate the significance of ß- hemoglobin reduction in
Schwochow et al. BMC Biotechnology 2012, 12:33 Page 11 of 12
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filtered compared to whole blood samples, we per- Received: 25 January 2012 Accepted: 27 June 2012
formed both an ANOVA as well as a Students t-test. Published: 27 June 2012

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