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  • Title: RT-PCR detection of mRNA recovered from archival glass slide smears.
    Author: Akoury DA, Seo JJ, James CD, Zaki SR.
    Journal: Mod Pathol; 1993 Mar; 6(2):195-200. PubMed ID: 8483891.
    Abstract:
    This manuscript describes a protocol for the utilization of glass slide preparations of hematologic specimens for the recovery of mRNA that is of a quality suitable for the reverse transcription-polymerase chain reaction (RT-PCR) detection of specific gene expression. Total cellular RNA obtained from archival bone marrow aspirate smears of 23 leukemia patients, which had been stored for periods of time from 1 day to 34 mo, were extracted, and 1 to 2 micrograms of each were subjected to RT-PCR using primer pairs specific for the amplification of beta-actin cDNA. Three pairs of primers for the amplification of beta-actin cDNAs of 83,260, and 540 base pairs were used to evaluate the length of mRNA that could be analyzed; the results indicate the consistent amplification of cDNA for the short- and intermediate-sized fragments as revealed by ethidium bromide fluorescence of agarose gel-resolved PCR products. To address the utility of RT-PCR analysis towards the detection of mRNA associated with specific gene alterations in such specimens, a primer pair for amplification of the E2A-PBX1 fusion cDNA was used in PCRs of RT cDNAs for each of the 23 specimens, three of which were pre-B-cell acute lymphoblastic leukemias known to have the t(1;19) karyotype alteration resulting in the fusion of the E2A and PBX1 genes. Agarose gel electrophoresis analysis of the products of these RT-PCR amplifications revealed the amplification of the fusion gene cDNA in only those cases for which there was cytogenetic documentation of t(1;19); these results were confirmed by the Southern filter hybridization of an internal E2A-PBX1 oligonucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)
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