does the quality od cDNA interfer in qRTPCR - (Aug/15/2006 )
Hi,
I was doing qRTPCR one step, but then i had to change for two steps qRTPCR, here i had to do cDNA, the funny thing is that i can not reproduce my results that i got in one step, and even when i repeat the same qRTPCR i am not getting reproducibility, i think the problem can be the step when a do cDNA from RNA. i haven had experience doing it, do have any tip.
I am using ready to go from amersham to do my cDNA.
thanks for any help
I was doing qRTPCR one step, but then i had to change for two steps qRTPCR, here i had to do cDNA, the funny thing is that i can not reproduce my results that i got in one step, and even when i repeat the same qRTPCR i am not getting reproducibility, i think the problem can be the step when a do cDNA from RNA. i haven had experience doing it, do have any tip.
I am using ready to go from amersham to do my cDNA.
thanks for any help
I have the same experience! In my case it was probably the quality of the RNA that gave the problems (I cannot do a nanodrop with my samples). There were a couple of samples that gave different levels for the same gene in the same pcr. For instance, I did a 18S and put all the samples 3x on it (same master mix, same volumes, same run etc) and most of the samples gave 3 extact same levels, but others not, and it could vary up to a factor 10000 difference.
I made new cDNA for those samples, but that did not resolve the problem, so it was probably the quality of my RNA.
I have the same experience! In my case it was probably the quality of the RNA that gave the problems (I cannot do a nanodrop with my samples). There were a couple of samples that gave different levels for the same gene in the same pcr. For instance, I did a 18S and put all the samples 3x on it (same master mix, same volumes, same run etc) and most of the samples gave 3 extact same levels, but others not, and it could vary up to a factor 10000 difference.
I made new cDNA for those samples, but that did not resolve the problem, so it was probably the quality of my RNA.
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Hi.
how do you check your quality of RNA? i was thinking the samething, probably the quality of my RNA is not good,so i started to measure diffrents concentrations of one RNA sample, what i´ve seem is that when it is more dilute, the OD is better, i mean, it can go until 1.9, which i was not getting before.
So my question is, which is the limit for dilution without damaging my RNA sample?
Second question, i get rip off of the DNA that is contaminating my RNA sample by adding, DNAse beffer and DNAse, then i incubated it for 15 minutes at 25 C, then i add EGTA and again i incubate for 10 minutes at 65 C.
Here my question, Do you know if it is posible to check, how clean is my RNA sample (if it is clean of all DNA contaminants), and should i repeat the same step twice in order to improve my quality of RNA free from DNA?
thanks