genomic DNA contamination in RT-PCR - (Oct/02/2008 )
I have been isolating total RNA from drosophila fly heads using the Trizol method, followed by use of Ambion's DNA free DNase kit. I used the suggested length of 30min Dnase incubation at 37C, followed by 2 min inactivation. I then directly proceed with an iscript reverse transcription reaction. I then use my cDNA preps for real time PCR with SYBR green. Although in most cases I designed intron spanning primers, in some cases this was not possible.
My problem: although I use the Dnase treatment, I have amplification in the no RT reactions (typically occurring 8-9 cycles later than the reaction in cDNA reactions)(I ran the rxns on a gel, with a specific band showing for these rxns).
I read that it is ok to use results from the experiment if the noRT amplification occurs 5 cycles or later than the cDNArxn. Is this correct? Should I treat my RNA preps for longer with the DNase without degrading the RNA?
My problem: although I use the Dnase treatment, I have amplification in the no RT reactions (typically occurring 8-9 cycles later than the reaction in cDNA reactions)(I ran the rxns on a gel, with a specific band showing for these rxns).
I read that it is ok to use results from the experiment if the noRT amplification occurs 5 cycles or later than the cDNArxn. Is this correct? Should I treat my RNA preps for longer with the DNase without degrading the RNA?
My problem: although I use the Dnase treatment, I have amplification in the no RT reactions (typically occurring 8-9 cycles later than the reaction in cDNA reactions)(I ran the rxns on a gel, with a specific band showing for these rxns).
I read that it is ok to use results from the experiment if the noRT amplification occurs 5 cycles or later than the cDNArxn. Is this correct? Should I treat my RNA preps for longer with the DNase without degrading the RNA?
when the amplification in no RT samples occurs 5 cycles later it should mean that the amount of the target genomic DNA is about 32-fold less than the cDNA, if assuming that the amplification's efficiency is the same for both the amplicones (genomic and cDNA) and has a value of "2". This last occurrence is quite unlikely. If we admit an amplification's efficiency of 1.7, the amount f genomic amplicon is about 14-fold less than the one from cDNA. If I'm not wrong, in the first case you should have an overestimation of about 3%, in the second of about 7%. You have to decide if the error is low enough...
We use Trizol and DNAfree and never had any DNA contamination. But it is a habit in our lab to use only upper 2/3 of of aqueous phase after adding the chlorophorm to Trizol (phase separation), because the lower part contains lots of DNA.
Many people agrees that DNAfree works very good, so maybe you have so much DNA after isolation that it can't be digested all.
My problem: although I use the Dnase treatment, I have amplification in the no RT reactions (typically occurring 8-9 cycles later than the reaction in cDNA reactions)(I ran the rxns on a gel, with a specific band showing for these rxns).
I read that it is ok to use results from the experiment if the noRT amplification occurs 5 cycles or later than the cDNArxn. Is this correct? Should I treat my RNA preps for longer with the DNase without degrading the RNA?
It could be endogenous RT activity in the Taq you are using. Does your primer set cross an intron/exon border? If so then genomic amplification should produce a larger amplicon, which is easy to detect.
Where have you read that it is ok to use results from the experiment if the noRT amplification occurs 5 cycles or later than the cDNArxn.
As i am also facing sam ekind of problem in my experiment.
It's possible to under digest DNA, but not possible to over digest it. Increase the DNAse digestion time and/or amount of DNAse.
Similarly, be sure you are not using too much template in your cDNA reactions. How many cells are you using for your RNA isolation? This can be lowered too, to decrease the amount of DNA you need to digest.