Evaluation and validation of total RNA extraction methods for microRNA expression analyses in formalin-fixed, paraffin-embedded tissues.

Author(s): Doleshal M, Magotra AA, Choudhury B, Cannon BD, Labourier E, Szafranska AE

Publication: J Mol Diagn, 2008, Vol. 10, Page 203-11

PubMed ID: 18403610 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 extraction method and block size on total RNA and microRNA yield and amplification success from FFPE blocks of 10 different tissues. The FFPE blocks used in this study were stored for up to 12 years.

   Summary of Findings:

   When RNA was extracted from lung and breast cancers using the RecoverAll and RNeasy kit there was a much higher RNA (2-3 fold) and microRNA (5-20 fold) yield than when the other kits were used. When RNA was isolated from 13 additional FFPE blocks that had been stored for up to 12 y, the RecoverAll kit produced a higher, purer and more consistent yields than the RNeasy kit, but there was no difference in the electropherograms. miR-24 and miR-103 were detected at slightly higher levels (2 fold) and miR-191 at comparable levels when RNA was extracted using the RecoverAll kit rather than the RNeasy kit. The 18S ribosomal RNA and hGUSB were also detected at higher levels (11.7 and 2.4 fold, respectively) when the RecoverAll kit was used, but due to variability between the tissues, the effect was not significant. On average, 1.65 fold less miRNA was detected in FFPE specimens extracted using the RecoverAll kit than in frozen specimens when extraction was performed with the mirVana kit. RNA yield increased linearly with section number for up to 12 sections, as long as additional proteinase K was added to sections with a cross-sectional area of >150 mm2. Finally, the RNA yield and CT using the RecoverAll kit was highly reproducible within a tissue and between operators.

   Biospecimens

   • Tissue - Cervix
   • Tissue - Breast
   • Tissue - Gall Bladder
   • Tissue - Lung
   • Tissue - Kidney
   • Tissue - Liver
   • Tissue - Stomach
   • Tissue - Uterus
   • Tissue - Prostate
   • Tissue - Spleen
   • Tissue - Testis

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic
   • Neoplastic - Normal Adjacent
   • Neoplastic - Carcinoma
   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	Spectrophotometry
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Analyte Extraction and Purification	Analyte isolation method	Rneasy FFPE kit Absolutely RNA FFPE kit High Pure FFPE RNA micro kit PureLink FFPE RNA isolation kit RecoverAll Total Nucleic Acid isolation kit mirVana miRNA isolation kit
   Biospecimen Aliquots and Components	Aliquot size/volume	<75 mm2 >150 mm2 4 sections 8 sections 12 sections 16 sections
   Storage	Storage duration	1-12 years
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-24 miR-103 miR-191 18S rRNA hGUSB

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