RNA from stabilized whole blood enables more comprehensive immune gene expression profiling compared to RNA from peripheral blood mononuclear cells.
Author(s): van der Sijde F, Li Y, Schraauwen R, de Koning W, van Eijck CHJ, Mustafa DAM
Publication: PLoS One, 2020, Vol. 15, Page e0235413
PubMed ID: 32589655 PubMed Review Paper? No
Purpose of Paper
This paper compared RNA yields and expression profiles between matched Tempus blood specimens and peripheral blood mononuclear cells (PBMC) from patients with pancreatic cancer. RNA expression was also compared in specimens collected prior to and after chemotherapy.
Conclusion of Paper
All PBMC specimens yielded sufficient RNA for Nanostring analysis after a single extraction but 10 of the 24 Tempus specimens required a second extraction to obtain sufficient RNA. Following normalization, the average count was higher in RNA from Tempus blood than PBMCs. Of the 730 genes investigated, 509 were detected in Tempus and PBMC specimens, 107 were detected only in Tempus specimens, and 6 detected only in PBMC. Of the genes found in both specimen types, 349 were differentially expressed with 192 higher and 157 lower in the Tempus than the PBMC specimen. Importantly, hierarchical clustering did not cluster any specimens by patient but instead perfectly segregated PBMC versus Tempus. Pathways analysis revealed significant differences between specimen types for all pathways with fewer of the defining genes found in PBMC than Tempus specimens. Cell type contribution as determined by gene expression found Tempus specimens contained more dendritic cells, CD45+ cells, mast cells, neutrophils, and natural killer cells and fewer cytotoxic cells, macrophages, B cells, and CD8+ T cells. Within the specimen types, there was a tendency to cluster based on timing relative to chemotherapy with only 2-3 post-chemotherapy specimens or each type misclustering with pre-chemotherapy specimens.
Studies
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Study Purpose
This study compared RNA yields and expression profiles between matched Tempus blood specimens and PBMC specimens from patients with pancreatic cancer. RNA expression was also compared in specimens collected prior to and after chemotherapy. Blood was collected into EDTA and Tempus tubes from 12 patients with pancreatic cancer the day before and after one cycle of chemotherapy. PBMCs were isolated from EDTA blood using LeucoSep tubes by the Ficoll-Paque method within 4 h of collection. PBMC were enumerated using a Countess II Automated Cell Counter, snap-frozen in liquid nitrogen, and then stored at -80°C. Blood in Tempus tubes was stored at -80°C within 4 h of blood collection. RNA was isolated from PBMC using the RNeasy Mini Isolation Kit and from blood in Tempus tubes using the NucleoSpin RNA Blood Isolation Kit. RNA concentration and integrity were evaluated using an Agilent 2100 BioAnalyzer. Levels of 730 immune related mRNAs and 40 housekeeping genes were quantified using the PanCancer Immune Profiling Panel on a nCounter Flex system. A threshold of 25 counts was considered positive for detection.
Summary of Findings:
All PBMC specimens yielded sufficient RNA for NanoString analysis after a single extraction but 10 of the 24 Tempus specimens required a second extraction to obtain sufficient RNA. After the second extraction, all specimens had enough total RNA for NanoString analysis and RNA of 300-4000 nt for further analysis. Thirty-one of the 40 house-keeping genes were selected for normalization of the 730 immune related genes. Following normalization, the average count was higher in RNA from Tempus blood than PBMCs (1188.64 versus 790.02, P<0.001). Of the 730 genes investigated, 509 were detected in Tempus and PBMC specimens, 107 were detected only in Tempus specimens, six were detected only in PBMC, and 108 were not detected in Tempus or PBMC specimens. Of the genes found in both specimen types, 349 were differentially expressed and a total of 192 had significantly higher expression in Tempus than the PBMC specimen (P<0.05) with 111 of these displaying ≥2-fold higher expression than in PBMC. Of the 157 genes with significantly higher expression in the PBMC than the Tempus specimen (P<0.05), 72 displayed ≥2-fold higher expression than in the Tempus specimen. Importantly, hierarchical clustering did not cluster any specimens by patient but instead perfectly segregated PBMC versus Tempus. Pathways analysis revealed significant differences between specimen types for all pathways with only antigen processing (P<0.001), B cell functions (P<0.001), cell cycle (P<0.001), cytotoxicity (P = 0.014), NK cell functions (P<0.001), and senescence (P<0.001) higher in PBMC specimens. In all pathways examined, fewer of the defining genes were found in PBMC than Tempus specimens. Cell type contribution as determined by gene expression found Tempus specimens contained more dendritic cells (P<0.001), CD45+ cells (P<0.001), mast cells (P<0.001), neutrophils (P<0.001), and natural killer cells (P=0.022) and fewer cytotoxic cells (P = 0.024), macrophages (P = 0.009), B cells (P<0.001), and CD8+ T cells (P<0.001). Within the specimen types, there was a tendency to cluster based on timing relative to chemotherapy with only 2-3 post-chemotherapy specimens or each type misclustering with pre-chemotherapy specimens.
Biospecimens
Preservative Types
- Other Preservative
- None (Fresh)
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA DNA microarray RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Tempus tube
EDTA tube
Biospecimen Preservation Type of fixation/preservation Tempus
EDTA
Biospecimen Aliquots and Components Blood and blood products Peripheral blood mononuclear cells
Whole blood
Biospecimen Acquisition Time of biospecimen collection 1 day prior to Chemotherapy
After Chemotherapy