Reverse transcriptase PCR

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Novice
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Reverse transcriptase PCR

 Hi everyone

Scientis solutions is very attractive place for begginers. Can anyone please help me in my RT-PCR issues? I am observing an RFU peak of 300 with ct=3.45 in one of samples for detection of viral genome. Can this be regarded as an amplification?
 

Ivan Delgado
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Hi Novice,

Hi Novice,

A Ct of 3.45 cannot be amplification. It is too early in the PCR to be real amplification. 

Novice
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 Thanx Ivan

 Thanx Ivan

I have been trying hard to detect dengue virus genome by using a consensus primer and LUX primers for each serotype. Even samples which are poitive for dengue using ELISA are not giving any results. After so many trials a ct of 3.45 was seen in one of the samples. 

I have used viral RNA extraction kit and MMLVRT for RT. cDNA when run on a gel has good yield with a smear also. Cant trace the issue why is not PCR working when theoratically primers are good and specific. Also have tried gradients and touchdowns with various conc of primer and template.

Any suggestions?  

Ivan Delgado
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Hi Novice,

Hi Novice,

I suggest using a commercial source of RNA as a control to make sure the PCR is working.