Hi,

Firstly, it's important to consider that each kit comes with its own recommendations. For instance, the Quantinova kit from Qiagen suggests using a cDNA template with a final concentration of ≤100 ng per reaction. On the other hand, the SSO Advanced kit from BIO-Rad recommends a cDNA final concentration ranging from 100 ng to 100 fg. For the SYBR Green PCR Master Mix and SYBR Green RT-PCR Reagents Kit from Applied Biosystems, the recommended final concentration is between 1 ng and 100 ng. Generally, a concentration of 100 ng works well for your reactions. This concentration pertains specifically to the template used in your reactions.

To evaluate the efficiency of your PCR amplification, you will need to create a calibration curve (a linear dynamic range). To determine this linear range, prepare a series of dilutions of your cDNA template, each diluted tenfold (with at least five different dilution points).

I strongly recommend adhering to the "MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments." (https://doi.org/10.1373/clinchem.2008.112797)

This guideline includes all the information required to conduct a successful qPCR assay. Additionally, following the manufacturer's instructions for your selected qPCR kit is the most effective approach to achieve success.

Best regards,

30ng to 100ng works fine usually. However you can adjust it according to your cdna concentration and purity.