This general Q-RTPCR protocol is summarized in Figure 1 and it can be easily adopted by any laboratory. It is a robust quantitative method for gene expression studies. We highly recommend constructing a standard curve for the gene of interest because it offers an accurate measurement of the mRNA without assuming equal efficiencies between the samples and the reference and the ease of obtaining standards by cloning the amplicon using a commercially available kit. Multiple housekeeping genes should be validated for normalization in each experiment and incorporation of an external standard (alien RNA) is also needed to identify possible PCR inhibitors between treatments and runs.

Figure 1. General laboratory procedure for Q-RTPCR using cloned standards and multiple housekeeping genes.

Acknowledgements
This work was partially supported by Kapoor Foundation funds, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo.

Competing Interests Statement
The authors declare no competing interests.

References

1. Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169-93, 2000.
2. Wong ML, Medrano JF. Real-time PCR for mRNA quantitation. Biotechniques 39:75-85, 2005.
3. Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23-39, 2002.
4. Suzuki T, Higgins PJ, Crawford DR. Control selection for RNA quantitation. Biotechniques 29:332-7, 2000.
5. Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, Grisar T, Igout A, Heinen E. Housekeeping genes as internal standards: use and limits. J Biotechnol 75:291-5, 1999.
6. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034, 2002.

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