The use of optimal cutting temperature compound can inhibit amplification by polymerase chain reaction.

Author(s): Turbett GR, Sellner LN

Publication: Diagn Mol Pathol, 1997, Vol. 6, Page 298-303

PubMed ID: 9458390 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine if PCR-based analysis is affected by OCT embedding medium. A single tumor specimen was divided and preserved with or without OCT prior to freezing at -20 degrees C. The effects of intentional contamination of purified DNA and RNA with diluted OCT (0-10%) and the efficacy of different purification methods were also investigated.

   Summary of Findings:

   PCR amplification of large gene products (530-763 bp) was unsuccessful for all specimens embedded in OCT prior to freezing, while specimens frozen in the absence of media had a 100% success rate for all amplicon sizes. PCR amplification of DNA extracted from OCT-embedded specimens was dependent on amplicon size as successful amplification of 96-280 bp gene products was observed. Washing OCT-embedded specimens prior to DNA extraction did not improve PCR success rate, although controls indicated that the saline wash was of no detriment. Intentional contamination of purified DNA extracted from control specimens resulted in failed amplification of the 763 bp product at a final OCT concentration of 5% or greater, with clear and appropriate amplification at OCT concentrations of 0-4%. PCR success rates remained unaffected by the extraction methods investigated, but were adversely affected by digestion (incubation) durations of 2 h or longer for OCT-embedded specimens or 24 h for specimens frozen in the absence of media, samples that also showed evidence of DNA degradation. RT-PCR analysis was successful for all specimens (with and without OCT) for the 346 bp amplicon. Successful RT-PCR analysis was inhibited by intentional OCT contamination of purified RNA at a final OCT concentration of 1%.

   Biospecimens

   • Tissue - Small Bowel

   Preservative Types

   • OCT
   • Frozen

   Diagnoses:

   • Neoplastic - Lymphoma

   Platform:

   Analyte	Technology Platform
   DNA	PCR
   DNA	Electrophoresis
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Frozen OCT
   Analyte Extraction and Purification	Analyte isolation method	Ethanol precipitation Phenol-chloroform extraction QIAamp tissue kit Instagene matrix
   PCR Specific	Length of gene fragment	96-763 bp
   RT-PCR Specific	Length of gene fragment	346 bp
   PCR Specific	Reaction solution	OCT (0-10%)
   RT-PCR Specific	Reaction solution	OCT (0-10%)
   Analyte Extraction and Purification	Incubation duration/condition	2-24 h

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