Hello!
I ran my first qPCR but I forgot to dilute the cDNA before running. I started with rtPCR of 2000ng of RNA in 20ul reaction volume. My RNA purity was great and I included a RNAse inhibitor.
I believe this gives me (1:1 ratio) 100ng/ul of cDNA.
I used 2ul of cDNA +13ul of my syberGreen primer reaction (15ul total volume). I ran the reaction with duplicates for each of my samples. The volumes and ratio described is based on my lab's internal protocol.
Nonetheless, I got Ct for my housekeeping gene (actin) of ~16-18 values between samples and my gene of interest with Ct values
1. yes, qRT-PCR is a highly sensitive technique, so high cDNA concentration will saturate the reaction. You may catch the difference window between the sample. So dilution is much needed.
2. Primer amount and concentration are two different things, ideally between 100 nM to 600 nM of FP/RP works fine.
3. Pipetting error
Hi
The range of Cq depends on the abundance of the transcript. This will determine how much diluting you need. By your early results I would say dilution of cDNA is not critical. Even if you wanted to dilute, only 2 fold would be enough.
After diluting 5 times, perhaps because of low abundance, you got results which were not acceptable.
Hi, I agree with Ali Javadmanesh , dilution in your case is not critical. For me - you should not to dilute your samples. It could improve your accuracy regarding your target gene.
When I want to estimare reaction effectiveness, I mix some samples with a lowest Ct (Cq) of my target gene. After that I try to dilute it in 5 fold at least 4 time. 2-fold it is too difficult for my hands ))) I mean accuracy of pipetting when adding in PCR tube.
Regarding primers concentration I always use 500 nM for start and can change it during reaction optimization. I agree with Saurabh Mandal , 100-600 nM is ok. But 500 nM more comfortable. In the case of self dimers (homodimers) you can prevent the insufficient of any primer in mix by adding a lot of each.
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