The Effects of Different Fixatives and Fixation Time on the Extraction and PCR Amplification of RNA from Paraffin Embedded Tissue
Abstract
A number of reports have indicated the theoretical possibility of performing PCR on RNA recovered from paraffin embedded tissue (PET). This would have broad ranging implications for research on archival human tissues from a variety of diseases. Although, it is established that RNA in PET has undergone significant degradation, the specific contributions of different fixatives and fixation times to this degradation are not known, A variety of mouse tissues were fixed immediately after sacrifice for either 2, 8 or 24 hours in either formalin, Omnifix II, or Carney's fixative. A fourth set of tissues was fixed in the same fixatives for 2 hours after a 24 hour postmortem interval. Spleen, muscle, liver and brain were processed and embedded in paraffin. RNA was extracted from deparaffinized cubes of tissue using a modified proprietary technique. Samples were quantitated and evaluated for purity spectrophotometrically. RNA was reverse transcribed using a random hexamer primed reaction. PCR amplification for cDNAs of the housekeeping genes glyceraldehyde-3-phosphatedehydrogenase (GAPDH) and hypoxanthine phosphoribosyl transferase (HPRT) was then performed. The PCR products were Southern blotted and probed with specific radio labelled oligonucleotides. The extraction technique developed provided consistent yields of RNA. GAPDH amplification indicated the presence of DNA contamination in the RNA preparations. Contamination was eliminated by brief DNase treatment, but only very limited evidence of intact GAPD HmRNA sequences was found. Amplifiable HPRT mRNA sequences were detected in 9/16 samples fixed in Omnifix II, 7/16 samples fixed in Carnoy's fixative and 3/16 formalin fixed samples. These samples were both DNase treated and untreated. Because of primer selection to preclude amplification of genomic HPRT, DNase treatment did not alter the results.
Document Details
- Document Type
- Technical Report
- Publication Date
- Apr 09, 1993
- Accession Number
- AD1011162
Entities
People
- Robert D. Foss
Organizations
- Uniformed Services University of the Health Sciences