A rapid real-time qRT-PCR assay for ovine beta-actin mRNA.
Beta-Actin mRNA is often used for normalization in gene expression experiments. We describe a sensitive, rapid and specific quantitative assay for the cytoplasmic ovine beta-actin mRNA. The assay was based on the polymerase chain reaction (PCR) with real-time fluorescence resonance energy transfer (FRET) measurements to amplify cDNA products reverse transcribed from mRNA. A part of the ovine beta-actin sequence was amplified from cDNA from fetal ovine synovial (FOS) cells with mRNA-specific primers and cloned into a plasmid clone. The assay standard curve was constructed with dilutions of this plasmid. The assay was linear over five orders of magnitude and detected down to 600 copies per reaction of target DNA. Intraassay coefficient of variation was 12%. Detection of the beta-actin gene was eliminated by designing FRET probes at splice junctions and detection of putative processed pseudogenes was minimized by using FRET assay design with four oligonucleotides. We measured 0.2 copies per cell in RNA preparations without reverse transcription and DNase digestion. This might represent processed pseudogenes. In constrast, we measured 1400 beta-actin mRNA copies per cell in RNA preparations after the RT and DNase steps. The assay should, therefore, be sensitive enough to measure beta-actin from a single individual cell. Dilution of target DNA in murine RNA or ovine cDNA preparations did not effect efficiency of PCR or linearity of the assay. The quantitative assay described in this work can be used to correct for variations in various real-time qRT-PCR experiments in ovine cells with diverse goals, including gene expression studies, quantitation of viral load in infected cells and in various gene therapy experiments measuring vector load and expression in transduced cells.
Bjarnadottir
Helga
H
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, IS-101 Reykjavik, Iceland.
Jonsson
Jon J
JJ
D000199:Actins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D002138:Calibration; D002478:Cells, Cultured; D003199:Computer Systems; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods* / Q000592:standards; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D051379:Mice; D041681:NIH 3T3 Cells; D012333:RNA, Messenger / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D012756:Sheep; D013582:Synovial Fluid / Q000378:metabolism*
D000199:Actins; D012333:RNA, Messenger