RNA QUANTITATIVE-ANALYSIS FROM FIXED AND PARAFFIN-EMBEDDED TISSUES - MEMBRANE HYBRIDIZATION AND CAPILLARY ELECTROPHORESIS

Fixed and paraffin-embedded tissues from pathology department archives are available for RNA expression analysis. We describe a general meth od for quantitation of specific RNA sequence extracted from single 6-8 -mu m human histological tissue sections cut from paraffin blocks. For each specific mRNA, the range of linear relationship between the log of the initial total RNA concentration and the log of the specific pro duct after reverse transcription (RT)-PCR must be established. We usua lly per form RT with avian myeloblastosis virus (AMV)-RT, using specif ic antisense primers and a variable number of cycles of PCR amplificat ion. The number of cycles must be adjusted within the range in which a linear relationship exists between the log of the amount of amplifica tion product and the number of cycles. The quantity of specific produc t is standardized relative to beta-actin mRNA to normalize for the deg ree of RNA degradation, which can be quite different among samples. Th e amplification products were quantified by dot blot and P-32-labeled hybridization probe or by capillary electrophoresis with a laser-induc ed fluorescence detector. The intratest variation range was for the do t blot mean +/- 10% standard deviation (SD) and for the capillary elec trophoresis mean +/- 3% SD.