What method(s) should be used for analyzing qPCR amplification products?

When using a validated primer, previous melt curve analysis (Tm values) can serve as a reference. If the Tm value is the same as observed in the past, it is reasonable to assume that the PCR product is the same as that obtained in the past. Keep in mind, however, that although identical PCR products will show the same Tm value, obtaining identical Tm values alone does not necessarily confirm [...]

What standard samples are recommended for preparing calibration curves?

Suitable standard samples are those that approximate the actual sample as closely as possible. For gene expression analysis studies, use cDNA prepared from samples collected under conditions in which the target gene is known to be expressed. For genomic analysis studies, use genomic DNA. Artificial standard samples (e.g., plasmid DNA) are not recommended. Even when the sequence of the amplicon [...]

Target gene expression levels are often normalized to the expression of a “housekeeping gene” (reference gene) to correct for differences in the amount of input RNA and variations in reaction efficiency. How do I select a suitable housekeeping gene?

There is no single, universally appropriate housekeeping gene suitable for accurate normalization in every experimental condition, as it is important to select housekeeping genes that do not vary in expression levels in the experimental system used. Common housekeeping genes that have been used in the past include GAPDH and ?-actin; however, reports in recent years indicate that expression of [...]

How can amplification of genomic DNA in total RNA samples be avoided?

To avoid amplification of genomic DNA in total RNA samples: *Design primers that avoid genomic amplification: select a large intron region and design forward and reverse primers in exons upstream and downstream of the intron. With this strategy, genomic amplifications cannot occur for large introns. When introns are small, genomic amplification can be differentiated based on differences in [...]

Which is a better starting sample for preparing RT-qPCR calibration curves, RNA or cDNA?

Calibration curves for RT-qPCR may be prepared by either of the following methods: *Serial dilution of RNA, followed by reverse transcription and real-time PCR *Serial dilution of cDNA (obtained by reverse transcription reaction), followed by real-time PCR Since the two methods evaluate different parameters, it is important to choose a method appropriate for the experimental system being used. [...]