Microarray analysis of RNA extracted from formalin-fixed, paraffin-embedded and matched fresh-frozen ovarian adenocarcinomas.

Author(s): Fedorowicz G, Guerrero S, Wu TD, Modrusan Z

Publication: BMC Med Genomics, 2009, Vol. 2, Page 23

PubMed ID: 19426511 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 formalin fixation and RNA extraction method on RNA integrity, PCR success, and microarray analysis in ovarian adenocarcinoma specimens. Matched FF and FFPE specimens fixed for 4-16 h at 4 degrees C or for 24 h at room temperature were used.

   Summary of Findings:

   RNA obtained from FF specimens had RINs between 6.5 and 8.5, and allowed for amplification of beta actin fragments approximately 800 bp long. In contrast, the RNA from FFPE specimens lacked the rRNA peaks necessary for RIN calculation. The maximum PCR fragment that could be successfully amplified from FFPE specimens was approximately 600 bp long. Although all RNA from FFPE specimens was degraded compared to FF specimens, specimens fixed at 4 degrees C for 4-16 h had RNA fragments over 500 bp long, but the specimens fixed for 24 h at room temperature had no RNA longer than 500 bp and no successful amplification. Microarray expression analysis showed a high degree of correlation (R2=0.743-0.837) between FF and FFPE specimens, but the scatter plots were tighter for FF than FFPE specimens. RNA extracted with the modified Ambion optimum FFPE RNA Isolation kit and with Agencourt FormaPure kit generally performed similarly.

   Biospecimens

   • Tissue - Ovary

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	RT-PCR
   RNA	DNA microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Biospecimen Preservation	Temperature of fixation/preservation	4 degrees C Room temperature
   Biospecimen Preservation	Time in fixative	4-16 h 24 h
   Analyte Extraction and Purification	Analyte isolation method	Modified Ambion optimum FFPE RNA isolation kit Agencourt FormaPure kit
   RT-PCR Specific	Length of gene fragment	200 bp 400 bp 600 bp 800 bp
   RT-PCR Specific	Targeted nucleic acid	Beta actin CLDN3

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