RNA quantitative analysis from fixed and paraffin-embedded tissues: membrane hybridization and capillary electrophoresis.

Author(s): Stanta G, Bonin S

Publication: Biotechniques, 1998, Vol. 24, Page 271-6

PubMed ID: 9494729 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare dot blot membrane hybridization and capillary electrophoresis as methods for relative semi-quantitative analysis of RT-PCR products. The following genes were examined: beta-actin, alpha-polymerase, epidermal growth factor, and c-erbB2. The tissues studied include normal liver, breast carcinoma, urinary bladder carcinoma, and cervix uteri dysplasia.

   Summary of Findings:

   Once a linear amplification range was identified for the gene of interest and the method of specimen preservation, RT-PCR analysis followed by dot blot hybridization or capillary electrophoresis successfully permitted quantitation of relative mRNA expression in both frozen and FFPE specimens. While both methods of quantification yielded reproducible results, intra-test variation was lower with capillary electrophoresis compared to dot blot membrane hybridization (3% vs. 10%). The two methods of quantitation produced results that were strongly correlated. Relative c-erbB2 mRNA expression, quantified by dot blot membrane hybridization of RT-PCR amplicons, was strongly correlated among frozen and FFPE colon adenocarcinoma specimens. Of note, the size range of amplicons was limited to between 74 and 97 bp.

   Biospecimens

   • Tissue - Liver
   • Tissue - Bladder
   • Tissue - Cervix
   • Tissue - Breast

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Benign
   • Normal
   • Autopsy

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR
   DNA	Dot blot or slot blot
   DNA	Electrophoresis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Snap frozen
   RT-PCR Specific	Targeted nucleic acid	Beta-actin Alpha-polymerase Epidermal growth factor c-erbB2
   RT-PCR Specific	Technology platform	Dot blot membrane hybridization Capillary electrophoresis

   View more