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Investigating gene expression profiles of whole blood and peripheral blood mononuclear cells using multiple collection and processing methods.

Author(s): Gautam A, Donohue D, Hoke A, Miller SA, Srinivasan S, Sowe B, Detwiler L, Lynch J, Levangie M, Hammamieh R, Jett M

Publication: PLoS One, 2019, Vol. 14, Page e0225137

PubMed ID: 31809517 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared yield, integrity and expression in RNA isolated from PBMCs and blood that were collected in different tube types and processed and stored under different conditions. Blood was collected from 5 healthy donors in PAXgene blood RNA tubes, RNAgard blood tubes, BD Vacutainers containing CPT, Acid Citrate Dextrose Solution A (ACD-A) tubes and triplicate K₂EDTA blood Vacutainer tubes. All tubes were inverted 8 times. RNAgard and PAXgene tubes were frozen, CPT tubes were centrifuged using the manufacturers protocol to obtain PBMCs which were immediately suspended in Trizol and frozen. The EasySep Direct Human Total Lymphocyte Isolation Kit was used to process 1.5 mL ACD-A blood to PBMCs which were frozen in TRizol while the remaining sample was stored at 4°C. One EDTA tube was immediately placed at 4°C.  PBMCs were isolated from an EDTA tube using Lymphocyte Separation Medium (LSM), subsequently frozen in Trizol. RNA was isolated from the third EDTA tube using Leuko- LOCK Total RNA Isolation System. RNA was extracted from PBMCs using a Trizol LS-based method. RNA was extracted from PAXgene blood using the PAXgene blood miRNA kit, and from RNAgard blood using the PAXgene blood miRNA kit after precipitation with BioMaxi precipitation buffer. ACK Lysing Buffer was added to a subset of blood specimens collected in EDTA and ACD-A tubes before extraction using miRNeasy mini kit. RNA was quantified by spectrophotometer and RNA integrity was evaluated on a Tapestation. mRNA expression was determined using Agilent Technologies Human Gene Expression Arrays co-hybridized with commercially procured human reference RNA.

   Summary of Findings:

   Mean RNA yield was highest in blood collected and stored in ACD-A (31.1 µg) and EDTA (18.4 µg) tubes without lysis. Lysing the blood in EDTA or ACD-A tubes with ACK lysis buffer resulted in lower mean yields (0.9 µg and 0.2 µg, respectively). Comparatively, mean RNA yields were higher from blood collected and stored in RNAgard tubes than in PAXgene Blood RNA tubes (3.7 µg versus 1.7 µg). The highest mean yield of RNA from separated PBMCs was observed when isolated  from EDTA blood with LSM (5.9 µg), followed by CPT blood (2.4 µg) with gel centrifugation, and ACD-A blood separated using magnetic beads (2.3 µg), then PBMC separated from EDTA blood using LeukoLOCK (1.1 µg). Mean RIN values were between 8.2-8.8 for PBMCs regardless of separation method; 8.2 for PAXgene blood specimens; 7.6 and 7.8 for ACK-lysed blood collected in ACD-A and EDTA tubes, respectively, but <6 for unlysed and blood collected in RNAgard tubes. Despite high RIN values, the PBMC separation methods generally showed higher distance ratios and lower correlation ratios between replicates than whole blood methods. The highest correlation ratios and lowest distance ratios were observed for ACD blood (ACK lysed or unlysed), while the lowest correlations and highest distances observed were with magnetically separated PBMCs. Overall expression patterns were consistent across methods, but there were qualitative differences between whole blood and PBMCs. Analysis of the 50% most variable genes showed separate clustering of PBMCs and whole blood with some overlap between the PBMCS isolated from EDTA blood using leukolock (only refrigerated PBMC) and blood, but importantly did not show clustering based on patient. Further, when considering whole blood methods PAXgene blood and RNAgard specimens (frozen and preserved) clustered separately from the refrigerated ACD and EDTA specimens. Analysis of differentially expressed genes in specimens shipped frozen/preserved versus refrigerated showed considerable overlap between blood (PAXgene and RNAGard versus whole blood) and PBMCs (leukolock versus others). While no pathways were found to be significantly affected by shipping temperature, several pathways were found to differ significantly between frozen (RNAgard and PAXgene) and refrigerated (EDTA and ACD-A blood) specimens, with the most profound effects traced to hematological system development and function.  When comparisons were limited to the 1,000 most differentially expressed genes by P-value,33 genes were found to be differentially expressed between preserved/frozen and refrigerated specimens both in PBMC and whole blood.

   Biospecimens

   • Cell - White Cells
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • Frozen
   • Other Preservative
   • PAXgene

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	DNA microarray
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anticoagulant	Acid-citrate-dextrose EDTA
   Biospecimen Acquisition	Type of collection container/solution	EDTA tube ACD-A tube CPT tube PAXgene Blood RNA tube RNAGard Tube
   Biospecimen Aliquots and Components	Blood processing method	Lymphocyte Separation Medium (LSM) Leuko- LOCK CPT Gel centrifugation Magnetic separation
   Analyte Extraction and Purification	Analyte isolation method	TRIzol PAXgene Blood miRNA kit ACK Lysing buffer with RNeasy mini kit and TRIzol LS
   Biospecimen Aliquots and Components	Blood and blood products	Whole blood Peripheral blood mononuclear cells
   Storage	Storage temperature	4°C Frozen

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