Competitor mRNA fragments for quantitation of cytokine specific transcripts in cell lysates.
Synthetic RNAs (sRNAs) specific for four human cytokines were constructed and used as an exogenous internal standard in a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The sequences of the sRNA and the target mRNA were identical except for a duplication or deletion of approximately 100 nucleotides. The size difference between these two templates permitted easy electrophoretic separation of their PCR products. The sRNA has polyadenylated sequences at the 3' end and can be added directly either to a cell lysate before RNA purification or to a reverse transcription reaction. One pair of primers is used to amplify the internal standard and the target simultaneously, and the ratio of the two PCR products remains constant throughout the amplification. This technique can be applied to quantitate specific mRNA in as few as 10 cells when the exogenous control is added directly to cell lysates. This method is sensitive, accurate and adaptable for quantitation of other transcripts.
Kozbor
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D
Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107.
Hyjek
E
E
Wiaderkiewicz
R
R
Wang
Z
Z
Wang
M
M
Loh
E
E
D015152:Blotting, Northern; D002457:Cell Extracts / Q000737:chemistry; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000138:chemical synthesis; D015347:RNA Probes; D012333:RNA, Messenger / Q000096:biosynthesis*; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D002457:Cell Extracts; D016207:Cytokines; D015347:RNA Probes; D012333:RNA, Messenger; D012313:RNA; D012194:RNA-Directed DNA Polymerase