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S.K. Panguluri and S.S. Kakar
4. Any housekeeping gene like GAPDH or b-actin or 18 S rRNA primers can be used to generate a standard curve. The starting quantity of the samples in the standard curve can be give as 100% and the relative quantities of the unknown or test samples will be calculated in the final results (which are displayed under starting quantity for each sample including the standards). 5. In this case, the relative quantities of the control and the treatments were taken in an Excel file and the mean, standard error and p-values (student t-test) can be calculated from each sample triplicates to generate final bar diagrams. 6. The final graphs from both absolute quantities and relative quantities will have normalized quantity or normalized relative expressions on its y-axis, respectively (7). Here, the normalized values will be obtained by the division of absolute quantities or relative quantities of test by its corresponding absolute quantities or relative quantities of the housekeeping enzyme. 7. The best standard curve will have efficiency values from 90 to 100% (e.g., E = 100.6% in the standard curve reaction shown in Fig. 3a and 99.4% in Fig. 3b). Also the R2 value given below the standard graph (Fig. 3) should be from 0.9 to 1. If multiple PCRs with multiple plates are to be done, a separate standard curve should be in every plate set. 8. For normalization, the relative quantities or absolute quantities of the housekeeping gene for all the samples can be done once and can be used across all different primers to be used for the same samples. Sometimes, the PCR data will have a very bad standard curve. Even in this case, the data will have threshold cycle values (Ct values) for all samples. If there is a good standard
Fig. 3. Good and bad standard curves. Here, 1 ml of cDNA was taken for each dilution and a total of five different dilutions were used to plot standard curve by using the housekeeping enzyme GAPDH. The qRT-PCR analysis was done using Sybergreen in Bio-Rad iQ Cycler and standard curves were analyzed. The standard curved with efficiency (E ) more than 99% was considered as best standard curves. All the samples were run in triplicates.