I am doing SYBR green based qPCR for HA gene of influenza. I have two questions:
1. I did a challenge experiment twice and in the first time, my primer (amplify 120bp) worked nicely in different organs while when I repeated the experiment with the same virus dose and same conditions, I observed in a series of experiments that this primer did not give any signals while another primer (amplify 304bp) gave nice ones. The 120bp primer did not work when I reduced the annealing from 55 to 53 as well. Could it be the case that in the second experiment there was less virus due to bad storage (for instance) and hence the low cDNA could be only detected with a primer (second) that amplify larger segment? What might be the other causes?
2. Could I compare the CT values of two experiments that are identical except in that the primers used are different i.e amplify different segments in the virus?
For answer for question 1, Actually I used this primer (the non working one) for the first time in this experiment. I tried using it at 10, 5 and 2,5 pmole and it did not work as well.
For answer 2 I did not think it is RNA quality. since the same cDNA sample it works with the old primer nicely.
Thanks Add your answer
Dear Mohamed
Concerning the 1st question: I fear that the non-working primer set might have a wrong base. you said that this pair amplifies a smaller fragment than the other pair. Does the bigger fragment contain the sequence of the smaller? If yes, I would recommend you to sequence the bigger fragment and check for the presence of the smaller. By this way you can ensure that your primer set has no mistake.
2nd question: you can never compare Ct values from two independent runs, even using the same primers. You have to include a positive control or, better, a standard with a known copy number. Within an experiment you can compare the Ct values.
Regards
Eugénia
The point is that this primer worked for me before in the first experiment but totally not in the second one. That is why it is strange.
It is right that you should fix the condition, but what about if you have to scree virus titer in 8 organs at 5 time points in each. No way that you will use another plates
To titer the virus you have to have a quantification assay. So, either you use a calibration curve or a delta delta Ct approach. Are you able to prepare a virus extract with a known copy number of the virus?
I think before all, test your primer maybe frequent uses made it bad. you can do primer dilution again.
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