cDNA equalization for reverse transcription-polymerase chain reaction quantitation.
Reverse transcription coupled with the polymerase chain reaction has been increasingly utilized to study gene expression. However, most previously published quantitative techniques are limited by accurate initial RNA quantitation and do not account well for the relative efficiency of reverse transcription. We have developed a technique of labeling and quantitating the random-primed cDNA product of a reverse transcription reaction. Using the polymerase chain reaction in conjunction with template dilutions or with an internal competitive template, we show that by normalizing the cDNA input into the polymerase chain reaction, we get accurate quantitation of gene expression. We call this procedure cDNA equalization of reverse transcriptase-polymerase chain reaction. This method is ideal for small clinical samples where accurate quantitation of input RNA is difficult.
Kolls
J
J
Department of Medicine, LSU Medical Center, New Orleans, Louisiana 70112.
Deininger
P
P
Cohen
J C
JC
Larson
J
J
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis / Q000235:genetics*; D005069:Evaluation Studies as Topic; D015870:Gene Expression; D005821:Genetic Techniques; D006801:Humans; D016676:Macrophages, Alveolar / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D004247:DNA