Myc Quantitative Realtime Rtpcr

To optimize quantification of MYC mRNA expression, we developed a real-time quantitative RT-PCR assay based on TaqMan fluorescence methodology using 40 primary tumor and 7 normal brain samples. Isolation of total RNA and cDNA synthesis was performed as previously described.[9,23,24] Kinetic real-time PCR quan tification of hTERT mRNA was performed using an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Rotkreuz, Switzerland) as described previ ously.1-25-1 For each PCR run, a master mix (Applied Biosystems) was prepared containing 200 nM of each primer and 400 nM probe. Thirty nanograms cDNA per RT sample was added to a final volume of 25 pL PCR master mix. The thermal cycling conditions comprised an initial denaturation step at 95°C for 10 min, and 50 cycles at 95°C for 15 sec and 60°C for 1 min. Experiments were performed in triplicate for each data point. Each PCR run included the five points of the standard curve (serially diluted DAOY PNET cell line cDNAs), a no-template control, the calibrator cDNA, and patient cDNAs.

The precise amount of total RNA added to each reaction mix (based on absorbance) and its quality are difficult to assess. We therefore also quantified transcripts of the 18S rRNA housekeeping gene as endogenous control, and each sample was normalized on the basis of its 18S rRNA content. The relative expression of MYC mRNA was calculated using the comparative CT meth-od.[26] The amount of MYC, normalized to 18S rRNA and relative to the calibrator, is then given by 2— AACt, where AACt=ACt (sample) — ACT (calibrator), and ACT is the CT of the target gene (MYC) subtracted from the CT of the housekeeping gene (18S rRNA). PCR efficiencies for MYC and 18S rRNA were measured as previously described[27,28] and were found to be >95%. Thus, the AACt method, in which the efficiency of PCR amplification of the target gene must be approximately equal to that of the housekeeping gene, is applicable.

We validated the method on a series of PNET cell lines with known MYC mRNA expression. MYC mRNA

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook

Post a comment