Q-PCRQuantitative Competitive Polymerase Chain Reaction
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Quantitative real-time PCR (Q-PCR) was performed using an ABI Prism 7500 sequence-detection system (Applied Biosystems, Foster City, CA, USA) with SYBR Green Realtime PCR Master Mix (Toyobo, Osaka, Japan), under the manufacturer's instructions.
To validate gene expression, we randomly chose the upregulated genes HIF-1A, NF-[kappa]B, and BIRC3 and the downregulated gene Bcl2 to do the q-PCR. The specificity of q-PCR primers was confirmed by regular PCR (Additional file 3).
The Q-PCR experiment was performed in triplicate, with each reaction solution (20 [micro]L) containing 200 nM of each primer, 10 [micro]L of 2X SYBR Green Supermix (BioRad), and 5-30 ng of the DNA template.
Absolute Q-PCR. To generate the absolute quantitative standard curve for Q-PCR analysis on lentivirus copy number measurement, we used the PCR product of the mouse GUS gene and cloned it with the TA cloning vector (pTA[R] Easy Cloning Kit), which was purchased from Genomics BioSci and Tech (Taipei, Taiwan).
(a) Expression of Cadm1 in different developmental stages of mouse inner ear examined by Q-PCR. The bar graphs show quantification of the PCR results with each value representing the mean [+ or -] standard error.
Moreover, quantitative analysis using q-PCR revealed that HDF supplemented with 30 [micro]M RA suppressed mRNA expression of iNOS to 20.8% of TNF-[alpha] alone (P<0.05; Figure 3B).
In order to investigate the effect of mechanical injury on ECM metabolism, the ECM production of L929 fibroblast was detected using Western blot and q-PCR after 5333 [mu]strain.
The authors would also like to thank the Genetics and Molecular Biology Laboratory, Directorate of Poultry Research, Hyderabad, for giving them access to their Q-PCR facility.