Standard-curve competitive RT-PCR quantification of myogenic regulatory factors in chicken embryos

Abstract

The reverse transcription-polymerase chain reaction (RT-PCR) is the most sensitive method used to evaluate gene expression. Although many advances have been made since quantitative RT-PCR was first described, few reports deal with the mathematical bases of this technique. The aim of the present study was to develop and standardize a competitive PCR method using standard-curves to quantify transcripts of the myogenic regulatory factors MyoD, Myf-5, Myogenin and MRF4 in chicken embryos. Competitor cDNA molecules were constructed for each gene under study using deletion primers, which were designed to maintain the anchorage sites for the primers used to amplify target cDNAs. Standard-curves were prepared by co-amplification of different amounts of target cDNA with a constant amount of competitor. The content of specific mRNAs in embryo cDNAs was determined after PCR with a known amount of competitor and comparison to standard-curves. Transcripts of the housekeeping ß-actin gene were measured to normalize the results. As predicted by the model, most of the standard-curves showed a slope close to 1, while intercepts varied depending on the relative efficiency of competitor amplification. The sensitivity of the RT-PCR method permitted the detection of as few as 60 MyoD/Myf-5 molecules per reaction but approximately 600 molecules of MRF4/Myogenin mRNAS were necessary to produce a measurable signal. A coefficient of variation of 6 to 19% was estimated for the different genes analyzed (6 to 9 repetitions). The competitive RT-PCR assay described here is sensitive, precise and allows quantification of up to 9 transcripts from a single cDNA sample.

Changes in estrogen receptor alpha expression in the bursa of Fabricius during chick embryonic development

“…cDNA were synthesized at 42°C for 50 min followed by inactivation of SuperScript II by heating at 70°C for 15 min. To remove template mRNA, samples were treated with 1 U of RNaseH at 37°C for 30 min (Alvares et al . 2003).…”

EST analysis of mRNAs expressed during embryogenesis in Gallus gallus

“…Genes often expressed at comparatively low levels, such as myogenic regulators, are only found after exhaustive sequencing. Prior work had determined MyoD to be 100 times less abundant than β−actin in stages E21-E26 chicken embryos (Alvares et al, 2003). In the present study, eleven ESTs (0.47%) coding for β-actin were identified in the LB1 library.…”

Effect of growth hormone on fatty acid synthase gene expression in porcine adipose tissue cultures

“…The standard b-actin curve was obtained in a similar way by modifying the concentration of the native and competitor cDNAs to concentrations of 10 -4 fmol. A detailed description of the methodology used in our competitive RT-PCR assays has been described by Alvares et al (2003).…”

“…The higher efficiency of the competitor cDNA amplification is reflected in the negative value for the intercept (Alvares et al, 2003).…”