Shalini Dubey

You can, but it is not commonly done. Most people either have a standard curve set up on the RT-qPCR and using that to normalize otherwise you use a reference gene to normalize your CT values from your target gene. The difficultly with trying to measure cDNA is unless you have the Qubit ssDNA kit, you cant use the normal dsDNA kit to quantify and any spectrophotometer method (Nanodrop) cannot distinguish@ dNTPs, cDNA, RNA from the reading.

Almost nobody quantifies their cDNA, its a meaningless number. What people do, however, is standardize their cDNA input into their qPCR as the same amount everytime. Users can frequently get meaningful, significant data by diluting their cDNA 1/10 or 1/20, and using the same volume in all the gene targets they're testing.

Craig Silver and Shawn Krueger thank you for the answers. Also, request you to guide me about the selection of a dilution factor and acceptable variation in Ct values of reference gene among the cases and controls.

Shalini Dubey

Depending on what qPCR reagent you are using it should recommend a range. I usually load anywhere from 100 pg to 100 ng of cDNA

Hi Shalini Dubey

You can use oligreen ( https://www.thermofisher.com/order/catalog/product/O7582#/O7582 ) to measure your cDNA. In the first article you have the way to measure.

1) One-step quantification of single-stranded DNA in the presence of RNA using Oligreen in a real-time polymerase chain reaction thermocycler.

2) Housekeeping while brain's storming Validation of normalizing factors for gene expression studies in a murine model of traumatic brain injury

3) Normalizing RT-qPCR data: are we getting the right answers? An appraisal of normalization approaches and internal reference genes from a case study in the finfish Lates calcarifer.

4) Gene expression in human skeletal muscle: alternative normalization method and effect of repeated biopsies.

5) Combined speed endurance and endurance exercise amplify the exercise‐induced PGC‐1α and PDK4 mRNA response in trained human muscle

6) An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research.

Good reading,

Jacó