RT-PCR not working - (Jan/04/2007 )
dear all,
im currently facing a lot of troubles with my RT-PCR.
after RNA isolation i use oligo dT primer for 1st strand synthesis and then i use gene specific primers for amplification.
the problem is i donot get amplification at all. i changed annealing temp, Taq amt. primer amt, template amount, but no success. same GSPs i use at same conditions they give sharp bands with genomic DNA. my rna samples are quantified and even the cDNA i confirm 1st with universal primers which give single band product. then only i proceed with my gene specific primers.
kindly suggest something,
thanks,
gsg
I have the same problem, tried everything of the above said, suspected in the RNA quality 
or maybe primers
Are you sure your RT reaction has worked? I donno if the universal primers u have mentioned can also smplify genomic dna if there is contamination! Otherwise technically it is difficult to make out why gene specific primers do not work where as universal primers do. ( I suppose your primers i have checked with some positive control)
i would say it's your primers.
if there is someone else in your lab who does RT PCR, see if they are kind enough to lend a sample of their cDNA for your reaction. if you get a band with their cDNA, and not yours, then you've got a problem with either the RNA or the strand synthesis. if you don't get a band with their cDNA, you have a PCR problem, (primers, enzyme, temps, etc).
it helps to eliminate the suspects one by one.
V
you should always run the RNA out on a gel first to check the quality. i had one sample that gave excellent spec readings, but when i ran it out on a gel... not so good.
thanks for replying,
i always trate my RNA samples with DNase, inactivate the DNase after treatment, confirm the integrity of rna on gel and its quality by always using universal primers as a contrl.
what else could be the positive control to check my primers, i put them with genomic DNa , they show desired fragment size and the sequence is same as that of the reported sequence in the database
Hi all...
Having troubles with my RT-PCR.....
Did RNA extraction using Trizol from Invitrogen the Gel pictures of RNA extraction is attached. Iam extracting the RNA from post larvae of Penaues monodon. i did a RT-PCR with Oligo dT primer 18mer and followed a protocol like this.....
Denaturation of RNA at 65°C for 5min (5ug)
Annealing of Oligo dT primer temp 25°C and also did with 37°C for 10 min.
The RTase to act at 42°C for 60min.
Inactivation of RTase at 70°C for 10 min.
for cDNA i had used a positive and -ve sample in the +ve had added RTase and in -ve dint add RTase but when ran the gel both the samples gave me the same patter is it how v get cDNA picture????
well did to confirm the cDNA using primers and doing a PCR with both the +ve and -ve Samples and got not a band but a luks like a primer dimer in both the samples.
can anyone juz tell me wats happening in my RT...and is my RNA extraction RTcorrect
thankyou
Having troubles with my RT-PCR.....
Did RNA extraction using Trizol from Invitrogen the Gel pictures of RNA extraction is attached. Iam extracting the RNA from post larvae of Penaues monodon. i did a RT-PCR with Oligo dT primer 18mer and followed a protocol like this.....
Denaturation of RNA at 65°C for 5min (5ug)
Annealing of Oligo dT primer temp 25°C and also did with 37°C for 10 min.
The RTase to act at 42°C for 60min.
Inactivation of RTase at 70°C for 10 min.
for cDNA i had used a positive and -ve sample in the +ve had added RTase and in -ve dint add RTase but when ran the gel both the samples gave me the same patter is it how v get cDNA picture????
well did to confirm the cDNA using primers and doing a PCR with both the +ve and -ve Samples and got not a band but a luks like a primer dimer in both the samples.
can anyone juz tell me wats happening in my RT...and is my RNA extraction RTcorrect
thankyou
Anybody has a reply for this query please reply.......
thanks a lot.........
Hi Chimmie
Have you tried to run your cDNA in a gel without adding anything? Is that your second picture?
You can also run your RNA to see how much you are obtaining. Ideally, you should run with a RNA sample which successfully worked before. You should get smear proportional to the amount of your RNA and ul poured in the gel.
The fact that negative and positive control give you the same results indicate a potential contamination, check all your reagents and primers.
Actually i am isolating RNA from tomato fruits, can u please tell which mehod gives better yield.
Hi Chimmie
Have you tried to run your cDNA in a gel without adding anything? Is that your second picture?
You can also run your RNA to see how much you are obtaining. Ideally, you should run with a RNA sample which successfully worked before. You should get smear proportional to the amount of your RNA and ul poured in the gel.
The fact that negative and positive control give you the same results indicate a potential contamination, check all your reagents and primers.