Comparison of methods in the recovery of nucleic acids from archival formalin-fixed paraffin-embedded autopsy tissues.

Author(s): Okello JB, Zurek J, Devault AM, Kuch M, Okwi AL, Sewankambo NK, Bimenya GS, Poinar D, Poinar HN

Publication: Anal Biochem, 2010, Vol. 400, Page 110-7

PubMed ID: 20079706 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 extraction method and DNAse treatment on DNA and RNA yield and PCR success. 7 unspecified visceral FFPE tissues from autopsy were studied.

   Summary of Findings:

   Amplifiable DNA was successfully extracted by all methods tested, but yield and c-myc copies/uL were highest when DNA was extracted using the phenol-chloroform or the TrimGen Wax free methods. An order of magnitude less RNA was extracted than DNA and the RNA was fragmented to lengths of approximately 200 bp or less. Only the TrimGen Wax free, Ambion RecoverAll (DNA or RNA), and phenol-chloroform methods gave consistent RNA yields. While extraction with the TrimGen Wax free method resulted in the highest RNA yield, the resultant RNA was not amplified as readily as RNA extracted using the Ambion RecoverAll kit (highest) or phenol-chloroform. Nucleic acids extracted with phenol-chloroform had the highest level of PCR inhibition, but extraction with Ambion RecoverAll and TrimGen Wax free kits also led to significant PCR inhibition. The yield of RNA increased when the digestion time used with the Ambion RecoverAll kit was increased from 3 h to 48 h, but PCR inhibition from the resultant RNA also increased. B2M copy number was slightly higher in specimens treated with DNAse prior to reverse transcription, regardless of extraction method used.

   Biospecimens

   • Tissue - Other

   Preservative Types

   • Formalin

   Diagnoses:

   • Autopsy
   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Real-time qPCR
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Fluorometry
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	3-13 years
   Analyte Extraction and Purification	Analyte isolation method	Strategene Absolutely RNA FFPE kit Roche Highpure RNA micro kit Sigma GenElute Phenol-chloroform Ambion RecoverAll Ambion Tri-Reagent TrimGen Wax free RNA kit
   Analyte Extraction and Purification	Deparaffinization	Kit solution Xylene
   Analyte Extraction and Purification	Cell/tissue permeabilization	Enzymatic digest 3 h Enzymatic digest 48 h
   Real-time qPCR Specific	Targeted nucleic acid	c-myc
   RT-PCR Specific	Targeted nucleic acid	B2M

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