I am new to real time Pcr and i am doing delta-delta ct method for relative quantification of gene expression. i wish to know that is it necessary to use the same dilution of target gene and house keeping gene for relative quantification...keeping the different dilution constant for my treated and untreated samples
I keep my dilutions the same across genes and samples. I think that's the way to go. It will ensure consistency in your results and your reaction set-up will be less prone to mistakes and variability.
Why would you want to use different dillutions anyway?
because my target gene expression levels are low...so i can use a dilution of 1:20 only however my housekeeping gene at a dilution of 1:20 gives me a ct of 4 which i guess is unacceptable so can i need to use my housekeeping gene in a diln of 1000000 so that i get a ct of 20
OK. Remember to check your reaction efficiency across different template amounts. If your efficiency is close to 100%, than there should be no problem in using different dilutions for reference and target genes. However, you will have to always use the same dilutions.
- Badges
- Science topic
- Similar topics
- Cell Biology
- Methods