The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?

Author(s): Gilbert MT, Haselkorn T, Bunce M, Sanchez JJ, Lucas SB, Jewell LD, Van Marck E, Worobey M

Publication: PLoS One, 2007, Vol. 2, Page e537

PubMed ID: 17579711 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 isolation method on DNA and RNA yield, amplifiable yield of nuDNA, mtDNA, RNA, proviral DNA and viral RNA, DNA size ratio, and SNP detection by multiplex PCR microsequencing from FFPE and BFPE Kaposi's sarcoma, lymphoma, lung, and spleen specimens. The effects of deparaffinization, incubation at 98 degrees C prior to proteinase k digestion, lithium carbonate washing, proteinase k digestion buffer composition, proteinase k digestion temperature and duration, extraction method, taq based DNA repair, hot alkali treatment, and critical point drying were examined.

   Summary of Findings:

   There was no effect of deparaffinization or critical point drying protocols on any of the parameters tested. Incubation of specimens at 98 degrees C for 15 minutes prior to proteinase K digestion increased the amplification of HIV-1 by RT-PCR, the amount of amplifiable nuDNA, and decreased multiplex PCR with minisequencing success without altering DNA or RNA yield, quantity of amplifiable mtDNA or RNA, or DNA amplicon size ratio. Addition of glycine to the proteinase k digestion buffer significantly reduced the amount of amplifiable nuDNA and mtDNA. Increasing digestion time from 24 h to 48 h led to an increase in amplifiable nuDNA and RNA. The authors state that digestion beyond 48 h increased amplifiable yields in some specimens, but the effect was variable and some specimens showed decreased amplifiable yield with extended digestion. Digestion at 65 degrees C instead of 55 degrees C increased yield of amplifiable RNA and amplification of proviral DNA, but further increasing the incubation temperature to 75 or 85 degrees C led to DNA and RNA degradation. Incubation at 120 degrees C in buffer with a pH of 11.2 (or in 0.1 M NaOH) increased the amount of amplifiable DNA but decreased total DNA yield and increased RNA degradation compared to specimens incubated for 48 h with proteinase K at 65 degrees C. Extraction of DNA and RNA with QIAamp DNA extraction kit led to increased yield of amplifiable RNA and nuDNA, but did not affect the amplifiable DNA size ratio. Repairing the DNA by incubation with Taq did not affect the amplifiable DNA size ratio, but increased the quantity of amplifiable nuDNA. In the small number of specimens examined, there was no significant effect of lithium carbonate washing on BFPE specimens, but there was a nonsignificant decrease in DNA and RNA yield (47-58%, and 36-60% respectively).

   Biospecimens

   • Tissue - Lymph Node
   • Tissue - Spleen
   • Tissue - Lung

   Preservative Types

   • Formalin
   • Other Preservative

   Diagnoses:

   • Neoplastic - Lymphoma
   • Neoplastic - Sarcoma
   • AIDS/HIV-related

   Platform:

   Analyte	Technology Platform
   DNA	PCR
   DNA	Real-time qPCR
   DNA	SNP assay
   RNA	Real-time qRT-PCR
   RNA	RT-PCR
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Formalin (unbuffered) Bouin's fixative
   Analyte Extraction and Purification	Analyte isolation method	Inclusion of dehydration and critical point drying (several protocols) QIAamp extraction Phenol-chloroform extraction Inclusion of DNA repair with taq polymerase
   Analyte Extraction and Purification	Washing	LiCO3 wash None
   Analyte Extraction and Purification	Protein digestion	55 degrees C 65 degrees C 75 degrees C 85 degrees C Buffer containing 25 mM glycine Standard buffer Hot alkali treatment (varied pH and temperature) None (only hot alkali treatment), 1 h 6 h 12 h 24 h 48 h 72 h
   PCR Specific	Targeted nucleic acid	Amelogenin HIV gag gene
   PCR Specific	Length of gene fragment	106/112 212/218
   Real-time qRT-PCR Specific	Targeted nucleic acid	Mitochondrial DNA hypervariable region Amelogenin
   RT-PCR Specific	Targeted nucleic acid	Beta 2 microglobulin HIV-1
   Analyte Extraction and Purification	Deparaffinization	None Xylene Pentane

   View more