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Research note: methodology for high-quality RNA
extraction from poultry whole blood for further gene
expression analysis
a
a
a
J. L. Mewis , X. Sun , M. J. Zuidhof & L. L. Guan
a
a
Department of Agricultural, Food and Nutritional Science, University of Alberta,
Edmonton, AB, Canada T6G 2P5
Accepted author version posted online: 21 May 2014.Published online: 16 Jun 2014.
To cite this article: J. L. Mewis, X. Sun, M. J. Zuidhof & L. L. Guan (2014) Research note: methodology for high-quality
RNA extraction from poultry whole blood for further gene expression analysis, British Poultry Science, 55:2, 194-196, DOI:
10.1080/00071668.2014.888397
To link to this article: http://dx.doi.org/10.1080/00071668.2014.888397
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British Poultry Science, 2014
Vol. 55, No. 2, 194–196, http://dx.doi.org/10.1080/00071668.2014.888397
Research note: methodology for high-quality RNA extraction from poultry
whole blood for further gene expression analysis
J. L. MEWIS, X. SUN, M. J. ZUIDHOF,
AND
L. L. GUAN
Downloaded by [University of Alberta] at 07:36 19 June 2014
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada T6G 2P5
Abstract 1. There are no published methods for RNA isolation from avian whole blood where nucleated
red blood cells prevent the use of established mammalian protocols. The aim of this study was therefore
to develop a protocol for total RNA extraction using avian whole blood by defining the effect of anticoagulants and sample purification protocols on RNA yield and quality.
2. Blood collections from the cutaneous ulnar or medial metatarsal veins of birds yielded adequate blood
volume (2–3 ml) draws. The experiment was a 2 × 2 × 3 factorial arrangement of treatments, with two
levels of DNase (0 and TURBO DNA-free Kit), two levels of Cleanup (0 and RNeasy MinElute Cleanup
Kit), and three anticoagulants (no anticoagulant, EDTA, or sodium citrate).
3. RNA was isolated successfully by adding TRIzol LS to 0.25 ml of chicken whole blood at 3:1 ratio. From
0.125 ml of avian whole blood, 2–3 µg of RNA with RNA integrity number values of 7.75 was successfully
isolated with the TRIzol LS extraction and an RNeasy MinElute Cleanup Kit.
4. This reliable protocol can be used to extract high yield and quality of total RNA from a small amount of
whole blood.
INTRODUCTION
RNA is a highly unstable molecule easily degraded
by endogenous RNases, which are ubiquitous in the
environment (Becker et al., 2010). RNA isolated
from tissue and whole blood has been widely used
in gene expression research and it has been successfully extracted from whole blood in mammalian
species in many research trials using the PAXgene
Blood RNA Kit (PreAnalytiX, Hombrechtikon,
Switzerland) and the TRIzol LS Reagent (Life
Technologies, Burlington, Canada). Isolating RNA
from avian whole blood is challenging because of
nucleated red blood cells (RBCs) that increase DNA
and protein contamination. Recently, total RNA has
been successfully isolated from an iguana species
(Glaberman et al., 2008), and methods for RNA
isolation from sauropsid species were evaluated
(Chiari and Galtier, 2011). Although both studies
included species with nucleated RBC, to date there
was no methodology published for total RNA
extraction from avian species. Whether such methodology could be applied or adapted to obtain highquality RNA from avian species was not known.
Whole blood can be used to elucidate molecular mechanisms influencing traits of interest. To
support further gene expression research, the
objective of the current study was to develop a
protocol for high yield and quality of total RNA
from avian whole blood by defining suitable sample collection protocols (anticoagulant effects)
and sample purification protocols.
MATERIALS AND METHODS
The University of Alberta Animal Care and Use
Committee approved animal study and blood collection protocols. The experiment was conducted
as a 2 × 2 × 3 factorial arrangement of treatments
in duplicate, with two levels of DNase (0 and
TURBO DNA-free Kit; Life Technologies; DNase),
two levels of Cleanup (0 and RNeasy MinElute
Correspondence to: L.L. Guan, Department of Agricultural, Food and Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta,
Edmonton, AB, Canada, T6G 2P5. E-mail: [email protected]
Accepted for publication 18 December 2013.
© 2014 British Poultry Science Ltd
RNA EXTRACTION FROM WHOLE BLOOD
Cleanup Kit; Qiagen, Toronto, Canada; Cleanup)
and three anticoagulant treatments (no anticoagulant, EDTA or sodium citrate).
Downloaded by [University of Alberta] at 07:36 19 June 2014
Whole blood sampling
Ten single-comb White Leghorn pullets were individually identified by wing bands at 16 weeks of
age. Feathers were hand plucked from the blood
collection site and skin was aseptically prepared
using 70% isopropyl alcohol. In total, approximately 2 ml of whole blood was collected from
each bird into a syringe, of which 0.25 ml of whole
blood was distributed in duplicate to three collection tubes containing EDTA, sodium citrate or no
anticoagulant. Whole blood samples were diluted
in a 1:1 ratio with RNase/DNase free HyPure
Molecular Biology Grade Water (Thermo
Scientific, Waltham, MA, USA) prior to addition
of TRIzol LS in a 3:1 ratio of TRIzol LS to whole
blood. Homogenised TRIzol LS whole blood samples were transported to lab on dry ice.
RNA isolation and purification
Total RNA was extracted using a TRIzol LS protocol (Life Technologies) following manufacturer’s
recommendations. Briefly, after mixing with
TRIzol LS, the blood sample was transferred into
the Precellys Soft tissue homogenising CK14 –
2 ml Kit (Bertin Technologies, France), and
homogenised in the Precellys 24 Homogenizer
(Bertin Technologies) using the programme of
2800 g, 2 × 30 s, 10 s between.
Four purification treatments were applied to
raw isolated RNA samples: TURBO DNA-free Kit
(Life Technologies; DNase; n = 6), RNeasy
MinElute Cleanup Kit (Qiagen; Cleanup; n = 6),
RNeasy MinElute Cleanup Kit and TURBO DNAfree Kit (DNase and Cleanup; n = 6) and control
(no purification; n = 6). All of the RNA samples
were dissolved with a known volume of RNase/
DNase free H2O and were stored at −80°C post
extraction.
RNA quantity and quality assessment
The concentrations of isolated total RNA samples
were measured using the NanoDrop 1000
Spectrophotometer
(Thermo
Scientific,
Wilmington, DE, USA). The quality was assessed
using the Agilent 2100 Bioanalyzer (Agilent
Technologies, Mississauga, Canada) with an RNA
6000 Nano Kit in accordance with manufacturer
protocols.
Statistical analysis
The data were subjected to analysis of variance
using the MIXED procedure of SAS (version 9.2,
195
SAS Institute, Cary NC, USA). There were no
anticoagulant effects using the methodology
described (P > 0.05); the anticoagulant was therefore omitted as a main effect from the analysis,
but included as a random variable because inclusion reduced the Bayesian Information Criterion.
The effects of DNase and RNeasy Minute Cleanup
were analysed with a two-way analysis of variance.
Differences between least squares means were
determined with pairwise comparisons, and
reported as different where P < 0.05.
RESULTS AND DISCUSSION
Technology has advanced to allow the study of
genome wide gene expression, rather than studying one gene at a time using microarray analysis
(Madabusi et al., 2006) or RNA sequencing of the
transcriptome (RNA-seq) (Tarazona et al., 2011).
For an accurate representation of gene expression
at the time of sample collection, it is important to
isolate intact RNA. Due to the lack of literature on
extraction of RNA from chicken whole blood, we
developed a reliable protocol to extract total RNA
from avian whole blood based on existing protocols for total RNA extraction of mammalian whole
blood and tissues.
Whole blood is a desirable sample for gene
expression for multiple reasons: blood collection
is easily performed, has minimal adverse side
effects and termination of the bird is not necessary. In addition, blood circulates throughout the
body and is therefore a good system wide representation of physiological status. According to
Pahl and Brune (2002), differences in techniques
used for blood collection and preparation of samples may cause changes in gene expression ex vivo.
In this study, adequate blood volumes (2 ml) were
successfully drawn from the cutaneous ulnar and
medial metatarsal vein with minimal haematoma
formation. Coagulation of blood samples may
occur if an anticoagulant is not used; however,
anticoagulants minimally preserve and stabilise
RNA and can interfere with downstream applications such as polymerase chain reaction (PCR).
Our preliminary analysis demonstrated that there
were no anticoagulant effects on RNA yield or
integrity (P > 0.05; data not shown).
The highest concentration of RNA was
extracted from the control treatment (Table).
However, the RNA integrity number (RIN) was
zero. It is therefore likely that DNA contamination
and/or degradation of RNA in the control treatment resulted in the increased concentration
measurement. Of the applied purification treatments, the highest concentration of RNA was
obtained from the DNase-only treatment; however, the RNA was highly degraded (RIN 0.57)
and not acceptable for further downstream
196
J.L. MEWIS ET AL.
Table. Effect of DNase and cleanup treatments on the
quantity of RNA extracted and RNA integrity number (RIN)
values
DNase
treatment
Cleanup treatment
No
No
Yes
Yes
SEM
Source of variation
DNase
Cleanup
DNase × Cleanup
No
Yes
No
Yes
RNA extracted
(ng/μl)
78.0a
25.8b
57.2a
10.0b
10.2
RIN
0.00c
7.75a
0.57c
3.82b
0.82
Probability
0.0519
0.0415
<0.0001
<0.0001
0.7822
0.0082
Downloaded by [University of Alberta] at 07:36 19 June 2014
a,b,c
Within a column, values not sharing a common superscript letter are
significantly different (P ≤ 0.05).
applications (Table). The combined cleanup
treatment and DNase treatment yielded a low concentration of RNA (25.8 ng/μl), as well as
degraded RNA (RIN 3.82). A significant two-way
interaction was observed for the RIN. The RIN
was acceptable for further downstream applications only when the cleanup treatment was
applied alone for RNA purification (Table).
Although a lower concentration of RNA was
extracted, an acceptable RIN is necessary for
further RNA analysis.
For RNA to be an appropriate sample for
real-time PCR, and further downstream applications, the RNA sample must be of high quality,
free from contamination of DNA, nucleases and
other proteins, as well as salts, polysaccharides and
organic compounds (Bustin and Nolan, 2004).
The RIN is a value reported from 0 to 10 and is
an indicator of the degradation/intactness of the
RNA value; the higher the value the higher the
intactness of the RNA (Mueller et al., 2004).
Commonly, a minimum RIN value of 7 is required
for downstream applications such as gene expression using microarray or RNA-seq (Madabusi et al.,
2006). In addition, various chemicals such as salts
and phenols are used throughout the RNA isolation procedure, which also contaminate the RNA
samples and decrease quality of RNA. A combination of TRIzol LS Reagent and RNeasy MinElute
cleanup yielded adequate amounts (2–3 µg) of
high-quality RNA (RIN 7.75) from a small amount
(0.125 ml) of avian whole blood, which was suitable for subsequent gene expression research. This
technique is an easy and reliable protocol for
poultry researchers to isolate high-quality RNA in
sufficient quantities to identify potential molecular markers through gene expression using avian
whole blood samples.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the technical
assistance provided by the staff and students of the
University of Alberta Poultry Research Centre,
Edmonton, Alberta.
FUNDING
Financial support for this study was provided by
Alberta Livestock and Meat Agency, Edmonton,
Alberta [Grant number 2011F121R].
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