title: Quantitative RT-PCR for measuring gene expression.
abstract: Classical Northern blot analysis for measuring mRNA requires too many cells to be practical for cell sorting. Yet, measurement of gene expression in small subsets within a heterogeneous population of cells is often desired. The PCR in combination with prior reverse transcription (RT-PCR) of the mRNA of interest provides a means for measuring gene expression using as few as one cell. When RT-PCR is performed, the reliability of the data can be highly subjective due to the efficiency of both RT and PCR steps. This subjectivity can be eliminated by a technique for quantitating specific RNA molecules using an internal RNA competitive reference standard (RNA-CRS), which is identical to the sequence of interest except for a deletion of 80 bases. Here we illustrate a strategy for quantitative PCR using a RNA-CRS, synthesized solely using nonplasmid-based PCR techniques. The competitive reaction consists of a constant quantity of wild-type mRNA (from 100-1000 cells) added individually to tubes containing a serially decreasing amount of RNA-CRS. The RT-PCR is performed on these samples, then the resulting product is analyzed by gel electrophoresis and densitometry. The procedure for preparing the RNA-CRS and subsequent RT-PCR steps are described in detail.
authors.affiliation: Roswell Park Cancer Institute, Buffalo, NY.
authors.forename: M C
authors.initials: MC
authors.lastname: Riedy
authors.forename: E A
authors.initials: EA
authors.lastname: Timm
authors.forename: C C
authors.initials: CC
authors.lastname: Stewart
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D012015:Reference Standards; D019061:src-Family Kinases
chemical_list: D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C053102:proto-oncogene proteins c-fgr; D019061:src-Family Kinases