Impact of RNA degradation on gene expression profiles: assessment of different methods to reliably determine RNA quality.

Author(s): Copois V, Bibeau F, Bascoul-Mollevi C, Salvetat N, Chalbos P, Bareil C, Candeil L, Fraslon C, Conseiller E, Granci V, Mazière P, Kramar A, Ychou M, Pau B, Martineau P, Molina F, Del Rio M

Publication: J Biotechnol, 2007, Vol. 127, Page 549-59

PubMed ID: 16945445 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This paper performed a comparative assessment of four different techniques for determining RNA quality from surgically excised, snap frozen specimens, as well as assess cDNA microarray performance. The tissues employed originated in patients with liver-metastatic colorectal cancer and included normal colon, tumorous colon, and tumourous liver.

   Summary of Findings:

   The authors developed a novel method of RNA quality assessment (the RNA Quality Scale, RQS) based upon the presence and height of the several peaks identified by electropherogram analysis: (1) low molecular weight RNA; (2) 18S and 28S ribosomal RNA; (3) the area in between ribosomal peaks; (4) nuclear RNA. A comparison among RNA quality determination techniques revealed that the means associated with sample classification differed significantly among all methods examined. Further, specificity (the probability of detecting reliable RNA samples) was highest for the RQS, RNA integrity number (RIN), and degradometer (95-86%); while sensitivity (the probability of detecting unreliable RNA samples) was highest for the 28S/18S ratio and RQS (90-82%). Reliable RNA thresholds were also identified for each technique: 28S/18S ratio >1.63; Degradometer <8.7; RQS >3; RIN >7.8. The authors concluded that RIN, degradometer DegFact, and RQS values were the best predictors of RNA quality for reliable microarray performance. These evaluation methods, combined with a post-hybridization clustering analysis of full chip expression ("dispersion tree"), provide stringent criteria upon which to identify samples that would give reliable or unreliable microarray results.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Normal Adjacent

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	DNA microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Automated electrophoresis/Bioanalyzer Specific	Quality metrics	28S/18S RNA ratio RNA Integrity Number (RIN) RNA Quality Scale (RQS) Degradometer DegFact

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