Maximizing RNA yield from archival renal tumors and optimizing gene expression analysis.

Author(s): Glenn ST, Head KL, Teh BT, Gross KW, Kim HL

Publication: J Biomol Screen, 2010, Vol. 15, Page 80-5

PubMed ID: 20008123 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of RNA isolation and quantification methods and real-time PCR parameters on RNA yield and subsequent amplification from FFPE renal carcinoma specimens. RNA was extracted from 3 sections of FFPE renal tumors from each of 10 patients.

   Summary of Findings:

   No difference in measured RNA yield was found when quantification was performed using RiboGreen rather than a Nanodrop spectrophotometer, and levels correlated highly with the beta actin (ACTB) cycle threshold (CT). The highest RNA yield was obtained using the MasterPure kit compared to the RecoverAll, High Pure FFPE, or ArrayGrade FFPE kits, but the yield was not significantly higher than when extraction was with Trizol. The RecoverAll and ArrayGrade kits yielded approximately 50% as much RNA as the MasterPure kit and the Highpure kit yielded less than 30% as much RNA. The RNA extracted using MasterPure kit was also free from DNA contamination, while RNA extracted using each of the other kits required a second DNAse treatment for one specimen, and 3 specimens required further treatment using the Trizol method. Extending the proteinase k digestion time from 3 h to overnight increased RNA yield when specimens were extracted with either the MasterPure kit, Trizol, or the RecoverAll kit. When reverse transcription was performed using gene-specific primers, the average CT was 2 lower than when reverse transcription was performed with random primers, but the relative abundance of the 23 assayed transcripts was not altered. Using custom TaqMan low density arrays instead of conventional 384 well plates, or a BioRad CFX 384 instead of an ABI 7900HT for real-time PCR, resulted in higher CTs which, in some cases, affected detectability.

   Biospecimens

   • Tissue - Kidney

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Fluorometry
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR
   RNA	Low density array

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	RecoverAll kit High Pure FFPE kit ArrayGrade FFPE kit MasterPure kit Xylene deparaffinization, proteinase k digestion and trizol extraction
   Analyte Extraction and Purification	Protein digestion	3 h Overnight
   Analyte Extraction and Purification	Nucleic acid digestion	Once Twice
   Spectrophotometry Specific	Technology platform	RiboGreen Nanodrop
   Real-time qRT-PCR Specific	Priming method	Random Gene-specific
   Real-time qRT-PCR Specific	Technology platform	ABI 7900 BioRad CFX384
   Real-time qRT-PCR Specific	Type of array	384 well plate TaqMan low density array

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