Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Ribonucleic acid extraction from archival formalin fixed paraffin embedded myocardial tissues for gene expression and pathogen detection.

Author(s): Sharma M, Mishra B, Vandana, Saikia UN, Bahl A, Ratho RK, Talwar KK

Publication: J Clin Lab Anal, 2012, Vol. 26, Page 279-85

PubMed ID: 22811362 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 method of RNA isolation from FFPE myocardial specimens of unspecified diagnosis on RNA yield, RT-PCR success and real-time qRT-PCR CT values. Specimens that were extracted using SDS or Trizol were deparaffinized in 37 degrees C xylene for 30 min while those extracted with RNeasy were deparaffinized for 15 sec in room temperature xylene and then centrifuged. Proteinase K digestion was at 60 degrees C for SDS (20 h) and Trizol (overnight) extracted specimens and at 55 degrees C for 15 min for RNeasy extracted specimens.

   Summary of Findings:

   RNA extraction was successful from all 16 FFPE specimens using RNeasy and 14/16 and 8/10 FFPE specimens using Trizol and SDS extraction methods, respectively. Further, significantly more RNA was obtained using RNeasy than using the Trizol (p=0.002) or SDS (p=0.012) methods. While glyceraldehye-3-phosphate dehydrogenase (GAPDH) and Rnase P could be amplified from all 16 RNeasy extracted specimens, GAPDH and Rnase P were amplified in 12 and 14 of 16 specimens extracted with Trizol, respectively. Only 1 of the SDS extracted specimens allowed for amplification of Rnase P, and GAPDH was not amplifiable from SDS extracted specimens. Further, CT values for RnaseP were slightly lower when specimens were extracted with RNeasy than Trizol or SDS.

   Biospecimens

   • Tissue - Heart

   Preservative Types

   • Formalin

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Trizol SDS RNeasy FFPE
   Analyte Extraction and Purification	Deparaffinization	37 degrees C xylene for 30 min Room temperature xylene for 15 sec than centrifuged
   Analyte Extraction and Purification	Protein digestion	Proteinase K at 60 degrees C overnight in Trizol Proteinase K at 60 degrees C for 20 h in TE SDS buffer Proteinase K at 55 degrees C for 15 min in buffer from RNeasy kit
   RT-PCR Specific	Targeted nucleic acid	GAPDH
   Real-time qRT-PCR Specific	Targeted nucleic acid	Rnase P

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