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1 PreAnalytiX (CH) c/o Becton Dickinson, Franklin Lakes, NJ 07417.
2 PreAnalytiX (CH) c/o QIAGEN GmbH, 40724 Hilden, Germany.
3 Becton Dickinson Technologies, Research Triangle Park, NC 27709.
4 Source Precision Medicine, Boulder, CO 80301.
aAddress correspondence to this author at Becton Dickinson MC 338, 1 Becton Drive, Franklin Lakes, NJ 07417. Fax 201-847-4851; e-mail lynne_rainen{at}bd.com.
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Abstract |
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Methods: We collected parallel blood samples from healthy donors into the new sample collection tubes and control EDTA tubes and performed serial RNA extractions on samples stored for 5 days at room temperature and for up to 90 days at 4 and 20 °C. Samples were analyzed by Northern blot analysis or reverse transcription-PCR (RT-PCR).
Results: Specific mRNA concentrations in blood stored in EDTA tubes at any temperature changed substantially, as determined by high-precision RT-PCR. These changes were eliminated or markedly reduced when whole blood was stored in PAXgene tubes. Loss of specific mRNAs, as measured by RT-PCR, reflected total RNA depletion as well as specific mRNA destruction demonstrated by Northern blot analysis. The salutary effects of PAXgene on mRNA stabilization extended to blood samples from eight unrelated donors.
Conclusions: Compared with whole blood collected in EDTA tubes and extracted by an organic method, the PAXgene Blood RNA System reduced RNA degradation and inhibited or eliminated gene induction in phlebotomy whole blood samples. Storage of whole blood samples in PAXgene tubes can be recommended for clinically related blood samples that will be analyzed for total or specific RNA content.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gene transcription analysis of blood samples is most often carried out on leukocytes isolated from phlebotomy samples (10). Whole blood is collected in various anticoagulants, including salts of citrate, heparin, and EDTA. These additives inhibit clotting but do little to maintain in vivo gene transcript quantities or control other preanalytical variables added by the cell preparation method. Therefore, accurate analysis of in vivo gene expression in whole blood may be complicated after phlebotomy by changes in cellular transcript patterns caused by sample collection, handling, storage, or uncontrolled coagulation (11). Intracellular RNA may be rapidly degraded ex vivo by specific and nonspecific endogenous nucleases (12). Furthermore, unintentional gene expression can be induced by phlebotomy, contact with foreign surfaces (13), or exposure to the contents of lysed cells, such as hemoglobin (14).
We developed the PAXgeneTM Blood RNA System (for research use only, not for use in diagnostic procedures), which includes a stabilizing additive in a blood collection tube, the PAXgene Blood RNA Tube (PAXgene), and a companion sample-processing reagent set, the PAXgene Blood RNA Kit, for purification of intracellular RNA from whole blood. Using agarose gel electrophoresis, Northern blot analysis, and quantitative real-time PCR, we compared the stability of total RNA and specific mRNAs in blood collected in PAXgene and EDTA tubes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For each study, we used PAXgene tubes containing a cationic detergent and additive salts (PreAnalytiX) and Becton Dickinson Vacutainer PLUSTM 6-mL dipotassium EDTA tubes to collect samples from each participant. Serial samples from individual donors were collected first in the EDTA tubes and then in the PAXgene tubes. All blood collection tubes were inverted five times for mixing immediately after collection and before storage at 22, 4, or -20 °C as indicated.
sample processing
RNA from whole blood collected in EDTA tubes was isolated by an organic extraction method (TRIzolTM LS; Life Technologies), according to the manufacturer’s instructions, and was further purified with a QIAampTM spin column (QIAGEN) and an on-column DNase digestion. We combined 1 mL of RNase-free water with 6 mL of Trizol LS reagent and 1 mL of blood in a screw-top centrifuge tube and mixed thoroughly. After chloroform (1.6 mL) was added to each tube, the contents were mixed again. Tubes were incubated and centrifuged, and the upper aqueous phase was removed and placed in a clean tube. Alcohol precipitates of the nucleic acids were resuspended in 240 µL of RNase-free water. We mixed the RNA samples with 840 µL of Buffer RLT (QIAamp RNA Blood Kit; QIAGEN) and 600 µL of absolute ethanol followed by mixing and brief centrifugation to remove liquid from the cap. The sample was applied in 700-µL aliquots to a QIAamp spin column and centrifuged for 1 min. Any remaining sample was used to repeat the filter wash. The QIAamp spin column was washed with 600 µL of RW1 wash buffer and centrifuged for 1 min. We added 80 µL of DNase digestion mixture [30 Kunitz units (15) of DNase dissolved in 10 µL of water plus 70 µL of buffer RDD] to the silica membrane, which was then incubated at room temperature for 15 min. The membrane was washed with 350 µL of RW1 buffer, centrifuged for 1 min, and washed twice more with 500 µL of RPE wash buffer, with centrifugation for 1 and 3 min between washes, respectively. RNA was eluted twice with 40 µL of RNase-free water.
For time zero (t0) controls, we extracted RNA from EDTA tubes within 30 min of phlebotomy and from the stored EDTA tubes at designated times by the TRIzol/QIAamp/DNase method described above.
For PAXgene tubes, we extracted RNA according to the manufacturer’s directions. PAXgene tubes were centrifuged for 10 min at 3000–5000g in a swing-out rotor. The supernatant was removed by decanting or pipetting, and 5 mL of RNase-free water was added to the pellet, which was resuspended by mixing and then centrifuged for 10 min at 3000–5000g. After the supernatant was removed, the pellet was resuspended in 360 µL of BR1 buffer by mixing. We added 300 µL of BR2 lysis buffer and then 40 µL of proteinase K solution, and the samples were digested for 10 min at 55 °C. Samples were then centrifuged for 3 min at maximum speed in a microcentrifuge, and the supernatant was transferred to a fresh 2-mL microcentrifuge tube, after which 350 µL of absolute ethanol was added to the tube and the sample was applied to the PAXgene Blood RNA System spin column. We centrifuged the spin columns for 1 min at maximum speed in a microcentrifuge and discarded the flow-through. Samples adsorbed to columns were digested with DNase (RNase-Free DNase Set; QIAGEN), and the column was washed with buffers supplied in the reagent set. RNA was eluted from the column twice with 40 µL of BR5 buffer, and eluates were incubated for 5 min at 65 °C in a heating block followed by immediate chilling on ice. PAXgene tubes were processed at designated times after phlebotomy by the PAXgene protocol. Extracted RNA from all samples was stored -70 °C.
sample analysis
RNA yield and purity.
Total RNA was determined by ultraviolet absorbance at 260 nm. We diluted 10 µL of the purified RNA with 90 µL of 10 mmol/L Tris-HCl, pH 7.5, and estimated the RNA concentration by absorbance (1 absorbance unit = 44 µg of RNA). We determined the purity of the isolated RNA by measuring the ratio of absorbance at 260 and 280 nm. RNA was also quantified by a fluorescence assay with RiboGreen (Molecular Probes) (16). RNA from each sample was assessed for purity by A260/280 ratios. To determine the integrity of rRNA, we analyzed 1 µL of eluate from each sample with the Agilent 2100 Bioanalyzer according to the manufacturer’s instructions.
Northern blot analysis.
Blood samples from different donors were collected into PAXgene and EDTA tubes. The total RNA from 2.5 mL of EDTA whole blood was immediately extracted by an organic extraction method (TRIzol LS) according to the manufacturer’s instructions. Total RNA from the corresponding PAXgene tubes was extracted according to the PAXgene protocol. We stored the remaining blood samples at room temperature and extracted RNA 1, 3, 5, and 7 days after phlebotomy.
We loaded 5 µg of total RNA from t0 samples onto a denaturing 1.2% agarose/formaldehyde gel. For all time points, constant volumes, corresponding to the volume of the t0 samples, were loaded on the gel. After electrophoresis, we transferred the RNA to a positively charged nylon membrane (Pall Corp.) by capillary transfer using 10x standard saline citrate (SSC) buffer (17). The nylon membrane was incubated at 80 °C for 10 min to immobilize the RNA. Bound RNA was hybridized with a radioactive probe, which contained complementary sequences of glyceraldehyde 3-phosphate dehydrogenase (GAPDH; GenBank accession no. X01677), IFN IEF SSP (GenBank accession no. L07633), or p53 (from the 53–6001 plasmid from Maxim Biotech). The hybridization was carried out overnight at 65 °C in the ULTRAhybTM hybridization buffer (Ambion). The nylon membrane was washed several times at 65 °C with washing buffers containing 2x SSC–1 g/L sodium dodecyl sulfate to 0.5x SSC–1 g/L sodium dodecyl sulfate. Probe radioactivity was visualized by exposing x-ray film to the nylon membrane at -70 °C for 6 h (GAPDH), 24 h (IFN IEF SSP), or 7 days (p53) with an intensifier screen.
Quantitative RT-PCR.
For reverse transcription, we used 0.2–1.0 µg of total RNA primed with random hexamers with MultiScribeTM (Applied Biosystems) reverse transcriptase, according to the manufacturer’s procedure. We assessed the quality of first-strand synthesis by PCR amplification of the first-strand product on an AB 7700 Sequence Detection System. PCR controls consisted of separate reactions in which either template or reverse transcriptase was eliminated. We did not observe any product in controls after 40 cycles of amplification.
The first-strand synthesis product was diluted in water and added to TaqManTM Universal PCR Master Mix (Applied Biosystems) together with Precision Profile primers (Source Precision Medicine); the total volume for PCR amplification was 25 µL. An initial 10-min (50 °C) incubation was followed by 40 cycles of PCR amplification (denaturation for 15 s at 95 °C and extension for 1 min at 60 °C). Assays are available from Source Precision Medicine.
PCR data were collected with Sequence Detection System software, Ver. 1.7. Results were exported into AB Relative Quantification software.
repeatability
We collected eight blood samples from a single donor into PAXgene tubes according to the instructions in the product circular. The RNA from the PAXgene tubes was isolated after 24 h of storage at room temperature, according to the PAXgene protocol, digested with DNase, and analyzed as described above. The GAPDH copy number was determined on 2 µL of each eluate with the GAPDH RT-PCR assay from PE Applied Biosystems.
calculations
Relative mRNA expression was determined by the 
CT method (18). PCR determinations for each gene in each sample were performed in quadruplicate. We eliminated replicates not meeting Source Precision Medicine quality-control standards for range and skew. The CT, or cycle threshold (19), is the cycle at which the PCR product crosses the detection threshold, usually at mid-log stage of PCR amplification. Each PCR reaction was multiplexed with specific primer probe sets for two genes in each well: the gene of interest and an endogenous control, 18S rRNA. rRNA was selected because it is highly resistant to nuclease degradation. The difference between the mRNA CT value and the 18S RNA CT value is CT:
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CT) represents the difference between CT values for a pair of conditions. Relative mRNA expression, assuming 100% PCR efficiency, is exponential and defined by the formula:
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CT was calculated by the method of Livak and Schmittgen (20). |
Results |
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). The total yield at t0, in µg RNA/mL of blood, varied from donor to donor, and the range of yields depended on the method of RNA isolation. Although the source of interindividual variability with respect to total RNA is unknown, it should be noted that in addition to leukocytes, reticulocytes contribute substantially to this RNA value, accounting for 1–4% of the erythrocytes in blood from healthy donors (21). Reticulocyte counts in healthy donors can thus range from 5 x 107/mL to 2 x 108/mL vs 7 x 106/mL for leukocytes. As the RNA yield varied by 2.7-fold among the donors in this study (and by as much as 5-fold in other studies not included here), the composite results shown in Fig. 1
are expressed in terms of the percentage of the initial RNA recovery.
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Total RNA was better preserved in PAXgene tubes than in EDTA tubes. After 5 days at 22 °C, mean RNA recovery for four donor samples stored in PAXgene tubes was 78%; in contrast, only 38% of the initial RNA was recovered from blood stored in EDTA tubes.
Storage under refrigerated or frozen conditions markedly extended RNA stability in PAXgene tubes. Typical results for a single donor’s blood stored in PAXgene and EDTA tubes at 4 and -20 °C are shown in Fig. 2
. PAXgene tubes stored at 4 °C for 30 days retained 100–120% of the initial RNA content vs 20% in EDTA. Frozen storage represented the best storage condition for PAXgene tubes. For all donors tested, the amount of RNA recovered from blood stored in PAXgene tubes was 
100% of the initial value after 30 days (Fig. 2
) and up to 90 days of storage at -20 °C (data not shown), whereas less than one-half of the initial RNA was recovered from blood stored frozen in EDTA tubes for the same duration (data not shown). At any temperature, blood stored in PAXgene tubes yielded markedly greater quantities of RNA than samples stored in EDTA.
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For the samples examined here, RNA purity, determined by A260/280 ratios, ranged from 1.8 to 2.1 in both EDTA and PAXgene samples, indicating that high-quality RNA could be obtained with both methods. The integrity of the rRNA in all samples was examined with the Agilent 2100 Bioanalyzer system. Shown in Fig. 3
are typical results of this analysis for two donor samples stored in EDTA or PAXgene tubes for 5 days at 22 °C. RNA was extracted at days 0, 3, and 5 after phlebotomy. For both donors, 28S and 18S rRNA bands were less pronounced at days 3 and 5 in the EDTA tube samples but remained clearly visible in the PAXgene samples.
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These effects of storage on gross RNA were reflected at the specific RNA level. Northern blots for three gene transcripts, GAPDH, IFN IEF SSP [an interferon (IFN)-
-activated gene], and p53, were performed on blood stored in either PAXgene or EDTA tubes at 22 °C for 7 days. Constant volumes, corresponding to the volumes of the t0 samples, were loaded on the gels for the Northern blot analysis to investigate the general stability of mRNA in blood samples. Loading equal masses for each time compensated for a general loss of mRNA in unstabilized blood samples. The results are shown in Fig. 4
. GAPDH transcript RNA was clearly detectable in PAXgene tubes for up to 7 days, although the band was substantially diminished at 7 days. In samples stored in EDTA tubes, the transcript was barely detectable at 3 days and undetectable at 5 and 7 days, indicating severe or complete degradation at these intervals. Similarly, IFN IEF SSP transcripts were detectable in samples stored in PAXgene tubes at 7 days but were barely detectable at 3 days in samples stored in EDTA and undetectable in these samples at 5 and 7 days.
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Northern blot analysis of the p53 gene showed that, in PAXgene tubes, the mRNA was preserved for 7 days at room temperature, although the intensity of the transcript band diminished slightly at 5 and 7 days of storage. In blood stored in EDTA tubes, however, the p53 gene transcript could be detected reliably only after 1 day of storage at room temperature. After 3 days of storage, the full-length mRNA band was only poorly detectable and was undetectable after 5 days of storage. As is evident from these results, PAXgene preserved specific RNA as well as total RNA. EDTA was without such beneficial effects.
The precision of molecular assays depends in large part on the repeatability of the sample collection and preparation methods and the quality of the resulting extracted nucleic acid. To test the precision of results, we examined the repeatability, expressed as the CV of the PAXgene Blood RNA System in three healthy donors. Total RNA yields per 2.5 mL of whole blood for donors 1, 2, and 3 were 16.12 ± 1.08, 13.59 ± 3.27, and 5.14 ± 0.46 µg, for CVs of 7%, 24%, and 9%, respectively. Because CT values are exponentially related to the copies of GAPDH measured, the %CV of CT values for GAPDH can be converted to the corresponding %CV for the copies of GAPDH, using an assumption of a perfectly efficient PCR amplification system. For GAPDH repeatability measurements, the mean (SD) was 19.24 ± 0.23 CT units, or 1% CV in terms of CT. The %CV for GAPDH copies measured is then equal to 2(±0.23), or 0.85–1.17 times the mean number (or 100%) of copies of GAPDH, for a 16% CV in repeatability of GAPDH copy number. These results are well within accepted values for such assays.
Relative expression for 36 mRNAs (Table 1
) was monitored in samples from two healthy donors by calculating the CT for each storage method relative to t0. The validity of this method was corroborated by the consistency of raw CT values obtained for mRNA and calibrator RNAs at t0. Samples processed immediately after collection in PAXgene and EDTA tubes exhibited minimal differences: CT values for interleukin-1ß (IL1B) were 28.5 and 30.0 for the PAXgene and EDTA tubes, respectively, whereas the corresponding 18S rRNA CT values were 13.5 and 15. The CT values were the same, indicating that the relative concentration of IL1B was initially identical in both tubes. Other mRNA measurements yielded similar results.
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With the method described above, 11 of 36 messages studied were undetectable in either PAXgene or EDTA blood from either donor (IL1A, IL2, IL4, IL6, IL7, IL-12p40, GM-CSF, TNFB, MMP1, MMP2, and CYCLD). One message, IL10, was detectable in donor A but not in donor B. Of the remaining 25 measurable mRNAs, expression for two transcripts, CRE and NFKB, was measurable and stable in EDTA and PAXgene blood from both donors.
Storage in EDTA tubes was accompanied by profound changes in specific mRNA expression, whereas PAXgene stabilized nearly all the species studied. Shown in Fig. 5
is the relative expression of 25 mRNAs in the blood of one donor (donor A) stored in EDTA (Fig. 5A
) and PAXgene (Fig. 5B
) tubes for up to 5 days at 22 °C. The t0 values for 18S and mRNA CT were comparable in both PAXgene and EDTA tubes. The data depicted in Fig. 5A
demonstrate that the relative amount of each of 22 messages changed in the EDTA tubes, because of either ex vivo gene induction or RNA down-regulation and/or degradation, by up to 100-fold. IL8, TNF, C-JUN, and FOS were induced within 4 h ex vivo in both donors; by 24 h, there was nearly 100 times more IL8 and C-JUN mRNA than the initial samples contained. By 24 h after phlebotomy, IFNG and ICAM were induced in donor A but not appreciably in donor B (data not shown). Relative amounts of 16 of 25 mRNAs (IL1B, IL10, IL15, IL18, TGFB, COX2, ICE, MMP9, HSP70, MYC, CYP2D6, STAT3, p53, BCL2, ENOS, and BAX) were markedly decreased in whole blood from one or both donors stored in EDTA tubes over the time period studied. Ninety percent of IL1B (donor B), IL10 (donor A), and IL18 (donor B) mRNAs were lost within 24 h after blood collection, whereas 99% of the original messenger content was lost for IL15, TGFB1, COX2, ICE, MMP9, HSP70, STAT3, p53, ENOS, and BAX after 3–5 days. From the lowest to highest amount of change, mRNA alterations ranged from a 99% decrease to a 100-fold induction. Such extreme changes in assayed mRNAs complicate the clinical relevance of gene product analysis in whole blood to the extent that clinical correlation is virtually impossible.
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The data presented in Fig. 5B
clearly demonstrate that PAXgene tubes better maintain the initial concentrations of individual mRNA species compared with EDTA tubes. None of the gene products studied was induced after 5 days of storage in PAXgene tubes. Furthermore, in PAXgene tubes, only three mRNAs showed degradation after 5 days, IL15, C-JUN, and IFNG, and even these three messages were stable for at least 24 h at 22 °C.
Unintended induction obscures degradation. This appears to be the case for IFNG and C-JUN in EDTA tubes (Fig. 5
, panel A vs panel B). All 16 of the mRNAs exhibiting degradation in EDTA tubes were clearly stable in PAXgene tubes. For donor B, with the exceptions of IL10, which was undetectable, and MYC, which decreased almost 10-fold after 5 days in EDTA tubes, results were the same as for donor A (data not shown). These results clearly demonstrate that RNA remains stable during medium-term storage at ambient temperature in PAXgene tubes and that the results of specific mRNA determinations can be used for correlative clinical studies.
Realistic shipping conditions often subject samples to 24-h transits at ambient temperature. The effects of such manipulations on gene activity are important to ascertain with regard to the clinical relevance of individual gene products. Ideal storage conditions maintain the concentrations of these products before and after shipment. To evaluate the suitability of PAXgene vs EDTA tubes in this application, we examined the concentrations of six mRNAs in samples from eight healthy individuals before and after storage at 22 °C for 24 h. The data represented in Fig. 6
depict the results of this study.
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In EDTA tubes, the means of relative gene expression for IL8, C-JUN, and CRE were 620-, 232-, and 10-fold higher, respectively, after 24 h of storage than for the corresponding t0 controls. By contrast, the average relative gene expression for transcripts stored in PAXgene tubes was 0.42–0.92 of the t0 concentrations. Clearly, for the gene products examined, the mRNA concentrations after storage in PAXgene much more closely approximated the initial values than the grossly induced concentrations (IL8, C-JUN, and CRE) in EDTA tubes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The purpose of this work was to determine the effect of blood storage conditions on gene activation and mRNA degradation. Our studies demonstrated that in unpreserved whole blood, ribosomal and mRNA is readily degraded. IFN IEF SS message is lost after 3 days of storage in EDTA, and clinically important genes, such as p53, are no longer detectable after 3 days. This indicates that traditional sample collection and storage tubes, such as EDTA tubes, may affect gene expression results of clinical studies by reporting falsely diminished quantities of important mRNA species. The appearance of first-response genes (e.g., p53) may be missed when mRNA is degraded; thus, important indicators of clinical conditions may be lost. Furthermore, it is impossible to make precise measurements of gene expression changes when specific mRNAs become undetectable, thus masking in vivo cellular responses. Constitutive gene products are used to calibrate the response of inducible genes; if these suffer degradation, the adaptive response of the gene of interest will be completely obscured. A detailed investigation of constitutive gene product degradation is beyond the scope of this report, however.
Induction of IL8 in EDTA samples (Fig. 6
) demonstrates one of the effects of handling and storage on blood after collection. IL8 induction suggests that cell activation occurs in the EDTA tubes before processing. Induction of IFNG, TNF, and ICAM over time was also observed, suggesting possible contact activation of cells in the EDTA collection tubes, which could lead to altered expression of genes involved in the proinflammatory process (11). Additional studies would be necessary to substantiate this hypothesis, but gene induction triggered by collection could greatly affect the conclusions of clinical gene expression studies.
In conclusion, these studies demonstrate that gene expression in whole blood is unstable over time when the samples are collected in EDTA tubes. Preservation in PAXgene tubes restricts ex vivo gene expression, allowing meaningful RNA assays and yielding transcript concentrations that are much closer to in vivo responses than can be obtained by other methods.
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Footnotes |
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References |
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therapy of competitive polymerase chain reaction. Blood 1996;87:1549-1555.. Blood 1994;84:3175-3181.C(T)) method. Methods 2001;25:402-408.[Web of Science][Medline]
[Order article via Infotrieve]
, IFN and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation. Cytokine 1992;4:239-232.[Web of Science][Medline]
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 U. Derhaschnig, R. Reiter, P. Knobl, M. Baumgartner, P. Keen, and B. Jilma Recombinant human activated protein C (rhAPC; drotrecogin alfa [activated]) has minimal effect on markers of coagulation, fibrinolysis, and inflammation in acute human endotoxemia Blood, September 15, 2003; 102(6): 2093 - 2098. [Abstract] [Full Text] [PDF]
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J.-M. Costa, A. Benachi, M. Olivi, Y. Dumez, M. Vidaud, and E. Gautier Fetal Expressed Gene Analysis in Maternal Blood: A New Tool for Noninvasive Study of the Fetus Clin. Chem., June 1, 2003; 49(6): 981 - 983. [Full Text] [PDF]
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) and EDTA (
) tubes for 5 days at 22 °C.
) and -20 °C (x) and EDTA tubes at 4 °C (
) and EDTA tubes (
).