Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Standardising RNA profiling based biomarker application in cancer-The need for robust control of technical variables.

Author(s): Stewart JP, Richman S, Maughan T, Lawler M, Dunne PD, Salto-Tellez M

Publication: Biochim Biophys Acta, 2017, Vol. 1868, Page 258-272

PubMed ID: 28549623 PubMed Review Paper? Yes

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This review on the effects preanalytical variability on RNA profiling examined the impact of block age on RNA yield, purity, and hybridization success and investigated the effects of microarray analytical workflow on the RNA profile.  The effects of block age were investigated in a total of 38 colon carcinoma specimens collected and processed between 1995 and 2007 by the Stratification in COloRecTal cancer (S:CORT) consortium, but no further details of specimen processing were described. RNA was extracted using the Roche High Pure RNA Paraffin kit. RNA yield and quality were determined by spectrophotometry. Actin 3’ to 5’ ratio and the assay success were determined following hybridization to the Almac Xcel array. Sequential sections from 42 FFPE colorectal tumor blocks were sent to two different facilities for RNA profiling. After extraction using the Roche HighPure RNA Paraffin kit, one facility used the Affymetrix 3’IVT PLUS reagents and the other used the NuGEN Ovation FFPE WTA System and Encore Biotin Module, after which, both facilities hybridized to Almac Xcel arrays.

   Summary of Findings:

   Tumor block age was not correlated with total RNA yield, purity (absorbance at 260/280 or 260/230), cRNA yield, or the 3’ to 5’ ratio of actin and specimens that were successful were not newer than those that failed. RNA profiles were strongly correlated when sequential sections were sent to different facilities that used different workflows before assay on Almac Xcel arrays (R=0.8293).

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	DNA microarray
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	Collected in 1995 Collected in 1996 Collected in 1997 Collected in 1998 Collected in 1999 Collected in 2000 Collected in 2001 Collected in 2002 Collected in 2003 Collected in 2004 Collected in 2005 Collected in 2006 Collected in 2007
   DNA microarray Specific	Template modification	Affymetrix 3’IVT PLUS reagents NuGEN Ovation FFPE WTA System and Encore Biotin Module

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