Towards Quantitative mRNA Analysis in Paraffin- Embedded Tissues Using Real-Time Reverse Transcriptase-Polymerase Chain Reaction A Methodological Study on Lymph Nodes from Melanoma Patients

Author(s): Abrahamsen HN, Steiniche T, Nexo E, Hamilton-Dutoit SJ, Sorensen BS

Publication: J Mol Diagn, 2003, Vol. 5, Page 34

PubMed ID: 12552078 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   To determine the effect of a delay after specimen acquisition and prior to freezing on the quantitative gene expression analysis of reference (beta-actin, beta2-microglobulin) and melanocyte-specific (MART-1) genes.

   Summary of Findings:

   A 24 h delay in biospecimen freezing resulted in a 50 and 64% decrease in expression of beta-actin and beta2-microglobulin, respectively, when compared to 0 h controls. While a large degree of variation was observed in MART-1 expression among tumors as a result of the delay in freezing, relative expression (to beta2-microglobulin) was comparable among groups and freezing delay durations.

   Biospecimens

   • Tissue - Lymph Node

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	1 h 2 h 3 h 24 h
   Real-time qRT-PCR Specific	Targeted nucleic acid	beta-actin beta 2-microglobulin MART-1

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2. Study Purpose

   To determine if the duration of formalin fixation adversely impacts the magnitude of gene expression as measured by real time qRT-PCR.

   Summary of Findings:

   Although beta-actin and MART-1 transcripts were detected in all samples, mRNA levels were significantly lower (85-99%) in samples formalin fixed for 1 day compared to paired frozen controls.

   Biospecimens

   • Tissue - Lymph Node

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Snap frozen
   Real-time qRT-PCR Specific	Targeted nucleic acid	beta-actin MART-1
   Biospecimen Preservation	Time in fixative	1 d 2 d 3 d 4 d 5 d 21 d

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3. Study Purpose

   To determine if amplicon size impacts resultant data from real time qRT-PCR analysis.

   Summary of Findings:

   Amplicon length affects resultant real time qRT-PCR data in a gene- and fixative-dependent manner. Data generated with short versus long amplicons was comparable when beta-actin was measured, but varied as much as 10-fold with MART-1 expression using fresh frozen tissue. In formalin fixed paraffin embedded samples, short amplicons increased expression 20- and 100-fold for beta-actin and MART-1, respectively, compared to long amplicon expression.

   Biospecimens

   • Tissue - Lymph Node

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Snap frozen
   Biospecimen Preservation	Time in fixative	5 days 21 days 1 day 2 days 3 days 4 days
   Real-time qRT-PCR Specific	Targeted nucleic acid	beta-actin MART-1
   Real-time qRT-PCR Specific	Length of gene fragment	97 bp 136 bp 313 bp 497 bp

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