A simple polymerase chain reaction method for detection and cloning of low-abundance transcripts.
A simplified polymerase chain reaction (PCR) technique for the detection, semiquantitation and cloning of low-abundance RNAs is described. This assay involves first-strand cDNA synthesis by reverse transcription of mRNA with specific oligonucleotide primers, followed by second-strand synthesis and PCR amplification, in the same tube with only the addition of TaqI DNA polymerase. The assay is sufficiently sensitive to detect target RNA from as little as 1 ng of total RNA. The beta-actin transcripts may also be simultaneously reverse transcribed, amplified and used as an internal standard to determine relative expression of specific RNAs. Using this simple technique, expression of the multidrug resistance (mdr 1) gene can easily be detected in human breast tumors. The technique is also applicable for the cloning of rare transcripts.
Fuqua
S A
SA
University of Texas Health, Science Center at San Antonio.
Fitzgerald
S D
SD
McGuire
W L
WL
D000199:Actins / Q000235:genetics; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics; D003001:Cloning, Molecular; D004351:Drug Resistance / Q000235:genetics; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000032:analysis*; D014158:Transcription, Genetic
D000199:Actins; D012313:RNA