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Difference in Ct value of replicates - (Oct/07/2012 )

Hello to all,
I am new to Q-PCR.
When I put Q-PCR first time for the newly synthesized cDNA, I did not get much difference in cT value between the replicates (of same sample) and also in housekeeping genes cT values of different samples. As I need to analyze the cDNA sample for four different primers, I freeze thawed cDNA for around 10 times and now I am getting difference in cT value (cT1 – 23; cT2 – 28 of same cDNA sample) and also in housekeeping genes cT value among different samples. I tried to reduce pippetting and manual error. Is it due to cDNA regradation? If so, why am I getting difference between cT values of the same sample? (I did duplicate for each sample). Please suggest me.



First, 5 cycle difference in Ct is indeed quite bad. The replicates variation is usually dependent on the value of the median Ct of replicates. In overall high Ct values (near 30), the variation is bigger than say in Ct 17.

The reason for high variation can be the pipetting (try pipeting the same amount of cDNA in a larger volume -> dilute), when talking about thawed cDNA, the incomplete dissolving can also play role, as well as not enough mixing of both template cDNA and mastermix before adding to well.
If the problem is consistent through several different samples, even fresh ones it may point to the machine (malfunctioning sensor or something) or plasticware problem (dirt or grease on the cover sheet, different evaporation from unsealed sheet, etc...).

You can test the thorough mixing and dissolving and increasing the volume together in next attempt, after run if the problem remains you can measure the remaining volume in all wells, that can tell you if you have a evaporation problem.

Also running triplicates instead of duplicates may be helpful.