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Gene expression differences between PAXgene and Tempus blood RNA tubes are highly reproducible between independent samples and biobanks.

Author(s): Skogholt AH, Ryeng E, Erlandsen SE, Skorpen F, Schønberg SA, Sætrom P

Publication: BMC Res Notes, 2017, Vol. 10, Page 136

PubMed ID: 28335817 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of collecting blood in PAXgene versus Tempus tubes and extracting RNA with the tube-specific protocol or a modified protocol on RNA yield and quality, and gene expression profile. For the first experiment, venous blood from four healthy females was drawn directly in PAXgene and Tempus tubes, inverted 10 times, and stored at -80˚C. For experiment 2, frozen blood from six healthy females in PAXgene tubes was obtained from the Norwegian Women and Cancer Cohort (NOWAC) and frozen blood from seven healthy females in Tempus tubes was obtained from Health Survey of North-Trøndelag. Experiment 3 included 41 specimens in PAXgene tubes from NOWAC and 20 specimens in Tempus tubes from HUNT3. Blood was thawed at room temperature for 16 h and then specimens for Experiments 1 and 2 were split for RNA extraction using the tube-specific protocol (PAXgene Blood RNA Tube protocol or the Tempus Spin RNA Isolation Kit) and the modified protocol. RNA from Experiment 3 was extracted using only the modified protocol which included PBS dilution, centrifugation, resuspension of the pellet in PAXgene kit buffer, proteinase K digestion in PAXgene buffer 2 for 10 min at 55˚C, filtration through a PAXgene spin column, addition of ethanol, filtration through a mirVana filter, filter washing and DNAse treatment, and elution in heated nuclease-free water. RNA yield and purity were determined spectrophotometrically. Integrity was determined by bioanalyzer. miRNA levels were assayed using TaqMan real-time PCR assays for miR-16, miR-181, and miR-423. Gene expression was determined by hybridizing Illumina TotalPrep RNA Amplification Kit amplified RNA to IlluminaHumanHT-12 v4 Expression BeadChips.

   Summary of Findings:

   Using the tube-specific RNA extraction protocol, RNA yields from blood in Tempus tubes showed a wide degree of variability but on average were higher than those from PAXgene tubes (0.98 ng/mL versus 0.55 ng/mL). When RNA was extracted using the same modified protocol for both tube types, RNA yield from PAXgene blood was comparable to that extracted with the PAXgene protocol (0.55 ng/mL versus 0.55 ng/mL), but the average yield from Tempus blood was lower than when RNA was extracted using the Tempus protocol (0.98 versus 0.69 ng/ml); however, this decrease was observed in just one of two experiments. Importantly, RIN was generally high and was higher when the modified protocol was used instead of the tube-specific protocol with the exception of blood in Tempus tubes in Experiment two.

   Levels of miR-16, miR-181, and miR-423 were higher when blood was collected in Tempus tubes than PAXgene tubes when RNA was extracted using the respective tube-specific protocol (P=0.01, P=0.008, and P=0.07, respectively) and when RNA was extracted from PAXgene tubes using the modified protocol than the PAXgene protocol (P<0.05, all). In contrast, miRNA levels in Tempus blood were unaffected by extraction method. 

   Although principle component analysis identified the largest difference in expression to be attributable to batch (experiment one versus experiments two and three), the second most important effect on the gene expression profile was collection tube system. Interestingly, extraction protocol, RIN, and RNA yield were not among the first six components. Up to 3143 probes differed significantly when extraction was by tube-specific methods in gene expression profiles of matched PAXgene and Tempus specimens from the same four individuals but significantly fewer probes differed significantly when RNA was extracted with the modified protocol (3143 versus 1540, P=0.00000046). For the other experiments that used both extraction methods, use of the modified protocol resulted in significantly fewer probes with significant differences between tube types than when tube-type specific protocols were used (1440 versus 2236, P<0.02). The overlap between the lists of probes that were differently expressed between tube types for the tube-type specific and modified protocols was 1066 in Experiment 1 (matched specimens) and 887 probes in experiment 2 (unmatched specimens), respectively. Importantly, the effects of tube-type were consistent in terms of direction, indicating the effect is due to the preservation system and not extraction method. Transcripts with higher levels in PAXgene specimens were on average slightly shorter and had a higher GC content then those preserved by Tempus. Transcripts from Tempus-preserved specimens were more likely to appear in the GO, KEGG, and REAC databases then those from PAXgene specimens. When lists of probes with significant differences between tube types in experiments 1 and 2 were compared, 708 probes were affected by the tube in the same way and three in the opposite way when RNA was extracted using kit-specific protocols and 417 were affected the same way and zero in the opposite way when RNA was extracted using the modified protocol. Further, 1199 were found to be affected the same way by tube type in experiment 1 compared to experiment 3 and 1330 probes in experiment 2 versus 3. The log-fold change between sampling systems for all probes was modestly to strongly correlated between experiments (r=0.60-0.86). Finally, the effects of sampling system were weakly correlated with those observed in a previous study using a different cohort.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative
   • PAXgene

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	DNA microarray
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	PAXgene tube Tempus tube
   Analyte Extraction and Purification	Analyte isolation method	Tube-specific protocol Modified protocol
   Biospecimen Preservation	Type of fixation/preservation	Tempus PAXgene

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