Nicole T. Vu

Hua Zhu

Edward D. Owuor

Mark E. Huggins

Vicky L. White

Arvind K. Chaturvedi

Dennis V. Canfield

James E. Whinnery

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

January 2004

Final Report

This document is available to the public

through the National Technical Information

Service, Springfield, Virginia 22161.

 

Office of Aerospace Medicine

Washington, DC 20591

Isolation of RNA From Peripheral 

Blood Cells: A Validation Study for 

Molecular Diagnostics by Microarray 

and Kinetic RT-PCR Assays  

Application in Aerospace Medicine

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NOTICE

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16. Abstract 

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17. Key Words 18. Distribution Statement 

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1

ISOLATION OF RNA FROM PERIPHERAL BLOOD CELLS: 

A VALIDATION STUDY FOR MOLECULAR DIAGNOSTICS BY MICROARRAY AND

KINETIC RT-PCR ASSAYS  APPLICATION IN AEROSPACE MEDICINE

INTRODUCTION

Extraction of cellular RNA is a critical step in the 

search for molecular information pertinent to gene 

expression. Expression profile studies in clinical diagnostic settings often involve samples collected in the 

field and at different time points, thereby requiring 

effective methods for collection, transportation, storage, and isolation of RNA to maintain the integrity of 

expression profiles. Human whole blood is a practical 

source of RNA for analysis of environmental exposure, 

performance evaluation, and pathogenesis of diseases 

because blood constituents maintain homeostasis, effect 

immunity and inflammation (1), or function as mediators 

in stress signaling (2-3) and in cellular communication of 

vascular associated tissues including those of the central 

nervous system (4-7). However, whole blood is a challenging tissue, since RNA information primarily resides 

in the small number of circulating blood leukocytes (8). 

Leukocytes comprise only 0.1% of the blood cellular 

fraction with approximately 5% of erythrocytes being 

in different stages of maturation and, therefore, having 

preformed mRNAs (9). Thus, isolation strategies using 

whole blood should recognize the limited quantities of 

useful RNA that must be protected from the hostile 

leukocyte ribonucleases, in addition to the abundance 

of preformed mRNA in reticulocytes, high protein content, and transcriptional activation of cells during sample 

processing in vitro. The challenges are how to preserve 

valid gene expression profiles from sample collection to 

isolation and how to maximize RNA yields and purity 

for sensitive downstream analyses. While different uses 

of nucleic acids have motivated many different ways for 

mRNA enrichment, the efficiency of these methods has 

not been determined for expression profiling of human 

whole blood. This report presents data demonstrating 

that the collection, treatment, and storage of collected 

blood samples can affect subsequent RNA isolation 

and analysis. Further, this report shows that total RNA 

isolated from human whole blood using the modified 

procedure of PAXgene Blood RNA reagent kit is suitable for gene expression analysis by cDNA microarray 

and kinetic RT-PCR assays.

MATERIALS AND METHODS

All reagents and supplies were sterile, nucleases free, 

and of molecular biology grade.

Sample Collection

Human whole blood was obtained from fully informed 

volunteer donors with the assistance of the Oklahoma 

Blood Institute (Oklahoma City, OK). Blood specimens 

were collected using a Safety-Lok Blood Collection Set 

(Becton Dickinson, Franklin Lakes, NJ) directly into 

PAXgene Blood RNA Tubes (Qiagen Inc., Valencia, 

CA) that contain a proprietary mixture of chemical additives for rupturing blood cells and stabilizing cellular 

RNA (10). The first 2-mL of blood was collected into an 

EDTA-coated Vacutainer Plus tube (Becton Dickinson, 

Franklin Lakes, NJ) as a discard sample. A total of 50-

mL whole blood was collected from each of the 4 donors 

using quantitative phlebotomy technique. Samples were 

gently mixed at room temperature (RT) on a hematology/

chemistry mixer (Fisher Scientific, Pittsburgh, PA) for at 

least 2 hours to ensure complete cell lysis and inactivation 

of ribonucleases prior to isolation of total RNA. 

To investigate the effects of storage temperature and 

aging of samples on RNA recovery, blood samples from 

the same subject were pooled into a single batch, and then 

each 18-mL portion of blood solution (≈ 5 mL whole 

blood + additive solution) was transferred to a sterile 

50-mL polypropylene tube (Becton Dickinson, Franklin 

Lakes, NJ). Sample aliquots were stored at either room 

temperature (RT) or 4°C for up to 10 days and were 

processed for RNA isolation at different time intervals 

(i.e., fresh, 24, 48, 72, 120, and 240 h).

Isolation of Total RNA from Human Whole Blood

Total RNA was isolated according to the PAXgeneTM 

protocol (11) with simple modifications. The nucleic 

acids were concentrated at 3000 × g in the Eppendorf 

5810R (Brinkmann Instruments Inc., Westbury, NY) for 

15 min with subsequent removal of the supernatant by 

aspiration. Each sample had 60 μL proteinase-K applied 

to it after the addition of standard volumes of suspension 

buffer (BR1) and binding buffer (BR2). Protein digestion 
2 

was carried out at 55°C for 20 min in a heat block with 

occasional vortex to ensure mixing. The digested mixture 

was centrifuged at 10,000 × g for 10 min to collect the 

supernatant. Following the separation of nucleic acids 

by affinity column, genomic DNA was digested with 

RNAse-free DNAse I directly on the column prior to 

elution. Contaminating salts and cellular impurities were 

removed by successive washing, and the RNA was eluted 

twice in 40 μL each of elution buffer (BR5). RNA yield 

and purity were first determined by UV scan (200–400 

nm) using Ultrospec 4300 Pro (Amersham Biosciences 

Corp., Piscataway, NJ), with RNA integrity evaluated 

by electrophoresing 10 μL of each RNA sample (≈

0.3-1.0 μg) from a denaturing gel-loading solution 

(formaldehyde/formamide/EtBr) on 1.2 % agarose gel in 

1× MOPS running buffer (pH 7.00). Electrophoresis was 

performed at 60 V (110 mA) for 1.5 h using the FB650 

DC power source (Fisher Biotech, Pittsburgh, PA). Images of the developed gels were acquired by the Typhoon 

9210 (Amersham Biosciences Corp., Piscataway, NJ) at 

a resolution of 100 μm and 600-V PMT. Selected RNA 

samples from the refrigerated group were next analyzed 

by RT-PCR and array hybridization.

Reverse Transcription and Quantitation in RealTime PCR (RT-QPCR)

To determine the performance of isolated total RNA 

in RT-PCR, aliquots of total RNA were amplified using specific primers corresponding to regions of human 

β-actin and cyclophilin genes, according to the protocol 

previously developed in our laboratory (12). Briefly, RT 

was performed with 0.6 μg total RNA and the MMLV 

reverse transcription system using random primers (Invitrogen, Carlsbad, CA). Platinum Taq DNA polymerase 

(Invitrogen, Carlsbad, CA) and Taqman® PDAR probe 

technology (Applied Biosystems, Foster City, CA) were 

employed in PCR assay of 5 μL RT products using the 

Smart Cycler (Cephied, Sunnyvale, CA) with real-time 

detection capability. Cyclophilin and β-actin were dually labeled with TET/BHQ-1 (Biosource International, 

Camarillo, CA) and FAM/TAMRA (Applied Biosystems, 

Foster City, CA), respectively. Plasmid DNAs with cloned 

PCR products of β-actin or cyclophilin were used to 

construct calibration curves for quantitation of the gene 

transcripts. The plasmids were prepared using pCRIITOPO vector and TOPO TA cloning technology (Invitrogen, Carlsbad, CA).

Microarray Hybridization

To investigate the effects of storage at 4°C on gene 

expression profiles, the total RNA isolated from blood 

samples of one subject was converted to fluorescent-labeled 

cDNA using the modified and optimized BD Atlas

Smart Fluorescent Probe Amplification protocol with 

random primers (13). Cy3-labeled cDNAs were generated 

for RNA isolated fresh and 72 h-aged blood samples, 

while Cy5-labeled cDNAs were generated for RNA 

isolated from blood samples after 24 and 240 h storage. 

The probes were hybridized, via dual labeling scheme to 

the Atlas Glass Trial Microarray (BD Biosciences, Palo 

Alto, CA) that includes 92 human gene targets and 8 

control targets. Images of hybridized arrays were obtained 

with the Typhoon 9210 Multi-Modal Imager (Amersham 

Biosciences Corp., Piscataway, NJ) at the set PMT of 

750 V. Image analysis was performed in ArrayVision

(Imaging Research Inc., St. Catharines, Canada) and 

SigmaPlot (SPSS Inc., Chicago, IL).

RESULTS AND DISCUSSIONS

RNA Isolation

Access to RNA requires both cell lysis and inactivation of cellular nucleases during isolation; hence, rigorous 

denaturing lysis conditions are imperative for obtaining 

intact RNA (14). Since human whole blood has a high 

protein content that can reduce extraction efficiency, 

protocols involving protease digestion should be employed to remove protein contaminants, in addition to 

increasing RNA yield with greater purity (15). Classical 

methods using organic solvents can adversely affect the 

yield and quality of RNA due to the presence of trace 

phenol and are relatively inefficient for recovery of minute 

RNA quantities in peripheral blood (16-18). The PAXgene protocol provides reagents for rapid stabilization 

of cellular RNA during sample collection, along with 

reagents for removal of hemoglobin and plasma proteins 

and, therefore, has potential for producing high quality 

RNA from blood. Preliminary investigation indicated that 

simple modifications to the PAXgene RNA isolation 

protocol, enabling 5-mL whole blood to be processed in 

a single tube, resulted in saving time and reagents, with 

no significant change in RNA recovery. The yield of RNA 

from peripheral blood leukocytes acutely depends on the 

physiologic state of the donor, reflecting the dynamic shift 

in circulating white blood cell fraction with subsequent 

wide-ranging variability on RNA constituents and their 

yields (9,19). Typically, the amount of RNA isolated in 

this study was 3 – 10 μg from 5-mL whole blood with 

a high purity ratio ranging from 1.9 to 2.1, except for 

RNA from samples that had passed 72 h storage at RT 

(Table 1). Illustrated in Figure 1 are changes in A260 over 

the elapsed time up to 10 d for samples stored at RT 

and 4°C. Although the entrapment of nucleic acids in 

a whitish film of lipids, proteins, or carbohydrates may 
3

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Elapsed Time (day)

0 2 4 6 8 10 12

Absorbance (A260)

0.0

0.2

0.4

0.6

0.8

1.0

RT

Refrigeration

Figure 1: Changes in absorbance A260 for RNA isolated from human whole blood 

stored in PAXgene Blood Tubes for up to 10 d at room temperature (RT) and 4°C 

(Refrigeration). RNAs from freshly collected blood samples were isolated on Day 0 and 

served as reference for all subsequent comparisons. Lines are median A260 values (n = 3).
4 

have contributed to low A260 values in some samples, 

the median A260 values for the refrigerated group were 

not significantly different from that for freshly isolated 

RNA samples (Friedman; p = 0.494). However, there 

was notable decline in A260 within the RT group, starting on the third d of storage, suggesting that peak RNA 

degradation occurred during this period at RT. Further 

analysis revealed that average RNA yields were considerably reduced by the 10th d (Dunnett’s; p < 0.05) and 

significantly different from average yields obtained from 

samples stored at 4°C (Bonferroni; p = 0.006). Accordingly, blood samples could be maintained for up to 72 h 

at RT in PAXgene reagents with negligible RNA loss; 

refrigeration delayed RNA breakdown for a much longer 

time. To be consistent with such effects, even though 

contrary to manufacturer’s information, it is therefore 

prudent to keep blood samples at 4°C if RNA isolation 

is not promptly performed following sample collection 

in the PAXgene system of preservatives. 

Following gel electrophoresis and depending on the 

tissue types, total RNA isolated from human tissues 

should produce a diffuse smear between 0.5 and 12 kb, 

with 2 bright 28S and 18S rRNA bands at approximately 

4.5 and 1.9 kb, respectively. The ratio of the intensities 

of 28S to 18S rRNA bands should be ≈ 2:1 (11,14,20). 

Displayed in Figure 2 is a typical image of molecular 

weight distribution on 1.2% agarose gel for RNA purified using the PAXgene system protocol. In examining 

electrophoresis data, the RNA samples exhibited banding 

patterns characteristic of RNA isolated from human blood. 

The 2 rRNA bands were within the expected ratio, no 

extra fragments were observed, and there were no signs 

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5

of genomic DNA contamination. As a result, it was judged 

that most samples were indeed composed of intact RNA 

and appeared to be comparable in quality, especially for 

samples stored at 4°C.

RT-PCR

The isolated RNA was tested by RT-PCR using primer 

pairs designed to amplify a segment of the β-actin or cyclophilin gene. Both β-actin and cyclophilin genes encode 

proteins that are essential for basal cellular activities and, 

therefore, are constitutively expressed in most cells (21-

23). Amplification was easily accomplished with 0.6 μg 

total RNA isolated from fresh and stored human blood. 

Concentrations of β-actin (number of actin molecules) at 

cycle threshold (Ct) were determined from the external 

calibration curve with log concentration values varied 

from 6.94 to 6.88, respectively, for fresh and 10-d aged 

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blood samples (Figure 3). The β-actin concentration 

ratio was 0.87, which was slightly biased toward fresh 

blood sample, though not considered significant. 

Cyclophilin in the same RNA samples produced 

similar results, with inconsequential changes in Ct

and log concentration values after 10 d storage. The 

resultant cyclophilin concentration ratio was 0.91, 

suggesting modest deviation in the number of cyclophilin mRNA transcripts that were isolated from 

fresh and stored blood samples. Although analysis 

by amplification is dependent on the relative abundance and the stability of the RNA molecule (24), 

nevertheless, the RT-PCR data, collectively with the 

electrophoresis and the spectrophotometric results, 

constitute an indication of negligible damage to total 

RNA during the 10 d storage at 4°C in PAXgene

reagent system. 
6 

Microarray Hybridization

The Ratio-Average Intensity (RI) plot for data points 

representing signals produced from intact RNAs on the 

tested cDNA microarray is illustrated in Figure 4. The 

intensity ratios for all array elements were based on volume 

intensity values, which had been background-subtracted, 

Cy3/Cy5-normalized, and artifact-removed using the analytical algorithm in ArrayVision. Further normalization 

based on average intensity for blank elements from the 

respective arrays allowed pair-wise comparison among 

the hybridized samples. The derived intensity ratios 

entailed relative expression levels and were plotted in 

SigmaPlot as a function of log-transformed mean 

intensity for each gene spot (25-26). Here, log intensity 

ratios for hybridized cDNA probes produced by 24-, 72-, 

and 240-h refrigerated blood samples (i.e., T1, T3, and 

T10, respectively) were established with a sample from 

freshly collected blood (T0) as a common reference. The 

convergence of data points to a tight line corresponding 

to intensity ratios of ≈ 1:1 indicates that signal intensities 

were essentially equivalent for these mRNAs, particularly 

data points representing extracted RNA from T1 blood 

sample. However, four genes (M65062, S62045, L34339, 

D13510) in the T1 and T10 samples deviated negatively 

from the reference sample as their log intensity ratios 

ranged from -0.973 to -1.539. Yet, T0 and T3 samples 

performed similarly in hybridization. This inconsistency 

in the observed data could reflect the fact that aging of 

samples was not the only factor that could contribute 

to the differential detection of these gene elements. 

It has been recognized that the Smart Fluorescent 

Probe Amplification using random primers maintains 

Geometric Mean Intensity

 [Log(T0*T1*T3*T10)

0.25]

5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2

Log Intensity Ratio

-2

-1

0

1

2

T3 v.s. T0

T10 v.s. T0

T1 v.s. T0

Figure 4: Reproducibility of mRNA profile by microarray hybridization analysis of selected 

gene targets using Atlas Glass Trial Microarrays. The data points for 96 array targets 

were correlated for all samples tested. cDNA probes were generated for RNA isolated from 

fresh (T0) and 72 h-stored blood (T3) samples by labeling with Cy3, while Cy5 was used to 

label cDNAs from 24 h (T1) and 240 h (T10) blood samples. Cy3- and Cy5-labeled probes 

were hybridized together on the same array. The optimized SMART Fluorescent Probe 

Amplification Protocol was employed using random primers.
7

representation of relative gene expression but can also introduce a slight ratio bias in hybridization signals (27-29). 

Alternatively, as was discussed in the study conducted in 

this laboratory (13), faithful replication of gene expression 

profiles was achieved with gene specific primers used in 

the probe synthesis procedure of the Smart protocol. 

Whereas disparity in hybridized signals remains to be 

verified by quantitative RT-PCR, preliminary analysis by 

Bonferroni procedure revealed insignificant differences 

(p > 0.05) between fresh and refrigerated blood samples 

at all time points. Thus, in spite of the fact that 4 % of 

the array elements were anti-correlated in a few instances 

of hybridization, the results from this study demonstrate 

acceptable reproducibility of the expression profiles using 

the PAXgene reagent systems and the described methods 

of sample collection and preservation. 

SUMMARY

Human whole blood is an essential source of nucleic 

acids for clinical investigation, since it is readily accessible 

and rich in molecular information. However, blood is a 

challenging tissue for RNA isolation and the establishment of a suitable method for collection of experimental 

samples. All of these procedural steps need to be carefully 

verified to ensure validity of the experimental data when 

using blood as a sample source. We evaluated a simple 

modification of the established protocol that provides a 

convenient and reliable method for the purification of 

RNA from fresh human whole blood. A real-time RTPCR assay was employed to determine RNA quality after 

isolation, along with microarray hybridization to assess 

the maintenance of gene expression patterns. The isolated 

total RNA using PAXgene system of reagents was of 

high purity and demonstrated excellent performance in 

RT-PCR and hybridization analyses. This report validated the use of PAXgene blood collection tubes and 

RNA isolation reagents for purification of RNA from 

limited amounts of fresh and stored whole blood collected in human performance studies, specifically in the 

examination of acute alcohol effects on performance. It 

is anticipated that this methodology will ultimately enable the characterization of molecular markers related 

to performance impairment for application in aerospace 

accident investigation and prevention.

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and Clinical Immunology, 6th ed. Los Altos, CA: 

Appleton & Lange.

2. D.L. Mann. 2003. Stress-activated cytokines and 

the heart: From adaptation to maladaptation. Annu 

Rev Physiol 65:81-101.

3. M.O. Kurrer, M. Kopf, J.M. Penninger, U. Eriksson. 2002. Cytokines that regulate autoimmune 

myocarditis. Swiss Med Wkly 132:408-13.

4. S. Rivest. 1999. What is the cellular source of 

prostaglandins in the brain in response to systemic 

inflammation? Facts and controversies. Molecular 

Psych 4:501-7. 

5. S. Laccroix , S. Rivest. 1998. Effect of acute systemic inflammatory response and cytokines on the 

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