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The Effect of Centrifugal Force in Quantification of Colorectal Cancer-Related mRNA in Plasma Using Targeted Sequencing.

Author(s): Xue VW, Ng SSM, Leung WW, Ma BBY, Cho WCS, Au TCC, Yu ACS, Tsang HFA, Wong SCC

Publication: Front Genet, 2018, Vol. 9, Page 165

PubMed ID: 29868115 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared mRNA quality, integrity, and expression profiles in plasma obtained by a single centrifugation at 3500 x g and that obtained by centrifugation at 1600 x g followed by 16000 x g. Blood from 18 healthy donors was collected into K₃EDTA tubes. Specimens were divided equally and plasma was obtained by centrifuging one part at 3500 x g for 10 min at 4˚C and from the other part at 1600 x g for 10 min at 4˚C followed by centrifugation of the supernatant at 16000 x g for 10 min at 4˚C. Trizol LS Reagent was added to the plasma and specimens were stored at -80˚C within 4 h of blood collection. RNA was extracted using chloroform and ethanol precipitation. The RNA was further purified using RNeasy MinElute Cleanup Kit. Eluted RNA was stored at -80˚C. RNA quality, size distribution, and yield were determined using a bioanalyzer. A sequencing library of colorectal cancer-related genes was prepared using a custom TruSeq Targeted RNA Expression Kit and sequenced on a MiSeq system.

   Summary of Findings:

   RNA extracted from plasma obtained by two-step centrifugation rather than a single centrifugation at 3500 x g had lower detection of both 18S and 28S rRNA (33.3% versus 88.9%), median RIN (1.15 versus 6.9, P<0.001), RNA concentration (100 versus 196.5 pg/µL, P<0.01), and median DV200 (41% versus 58.5%, P<0.01). Of the 108 genes sequenced, 69 were detectable and 16 undetectable, regardless of centrifugation protocol. The remaining 23 genes were found only in some specimens (1-5 of the 18) and occurred at low counts; however, 17 of these were identified only when centrifuged twice and six only when centrifuged once. The 25 genes with the highest expression levels were strongly correlated between plasma centrifuged at 3500 x g and that centrifuged at 1600 x g followed by 16000 x g (R2=0.9471, P<0.0001). Only two of these twenty-five genes, MYC and HIF1A, were found to be differentially expressed, with both having higher expression in specimens centrifuged twice (16.67 fold change, adjusted P=0.0025 and 5.56 fold change, adjusted P=0.0457, respectively). Importantly, clustering did not segregate specimens based on centrifugation protocol.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Next generation sequencing
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Centrifugation	Different number of centrifugation steps compared Multiple speeds compared
   Next generation sequencing Specific	Targeted nucleic acid	CRC-related genes

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