RT-PCR detection of mRNA recovered from archival glass slide smears.
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)
Akoury
D A
DA
Department of Pediatrics, Emory University SOM, Atlanta, Georgia.
Seo
J J
JJ
James
C D
CD
Zaki
S R
SR
D000199:Actins / Q000032:analysis; D000293:Adolescent; D001483:Base Sequence; D015139:Blotting, Southern; D001853:Bone Marrow / Q000737:chemistry / Q000473:pathology; D002648:Child; D002675:Child, Preschool; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005784:Gene Amplification; D015870:Gene Expression; D006405:Hematology / Q000379:methods; D006801:Humans; D007223:Infant; D007938:Leukemia / Q000235:genetics / Q000473:pathology; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*; D014178:Translocation, Genetic
D000199:Actins; D004273:DNA, Neoplasm; D009841:Oligonucleotides; D012333:RNA, Messenger