Getting signal in the negative controls - (Mar/28/2007 )
Hello ! I really need some help, please... I'm new doing real time PCR and I have never done PCR before.... So, I have almost no experience (only from the theory). I have done a few PCR experiments using RNeasy kit (Qiagene) for RNA extraction (from a cell line); GeneAmp kit (Applied Biosystems) for RT and LighCycler SYBR Green for PCR and this are my questions:
I. Are Trizol method better than RNeasy kit for avoids gDNA contamination (I have applied twice the DNase digestion step).
II. I'm getting a signal in my template free negative control (nuclease free water instead of template) and in the No RT controls as well. Should I think it is not gDNA contamination because the “No template control” gave me a signal too? (Somebody has suggested me the contamination could be in the primers... because I used the stock solution every time…I should have aliquotated them. If somebody has a different idea or advice, please give me a tip.
III. I have run the 4 negative controls (45 cycles) and the Crossing Points are as follow:
1- No RNA in RT rxn, CP: 34.25
2- No Reverse transcriptase in RT rxn, CP: 31.77
3- No template control, CP: 35.85
4- No amplification control, No CP.
Can I suspect there are contamination at two different levels because the No RNA has a CP higher than the No RT control ??
IV. Again, I don't have experience... Could somebody give me some useful advice about the most important things I need to be very careful with in real-time PCR (ie: set up the PCR away from where PCR reactions are opened) or suggest me some site that describes this topic.
My excuses for this so long consultation... Any help will be grateful !!
Thanks a lot !!
wellcome to the mysterious world of real-time PCR. Some things are allways strange but if you figure them out, this really is a great tool.
Your questions: RNeasy vs. Trizol - depends a little on the tissue you are using. I have no contamination problems with RNeasy even though I often drop the DNAse step - the important point is primer design here!
Your negative controls - the most important question: DID YOU CHECK THE PRODUCT ON A GEL??? The second important question: IF NOT, WHY? No, just kidding the question is - how are the melting curves looking?
In many cases, primer dimer and unspecific amplification will occur if you have enough cycles. SYBRgreen based methods are not template specific, so everything that forms a double strand will result in a signal. I have learned not to trust CP values above 30 for RT-PCR, as this is often unspecific amplification. But you can figure that out by looking at the melting curves and looking at the gel.
What are the CP values of your cDNA templates?
If these remarks don't help, please paste a picture of (I) the melting curves, (II) the gel and (III) the amplification curve.
Don't worry, you will sort it all out!
Krümel, about your question... YES, I have always checked the product on a gel. The melting curves for the controls have peaks in the same range as for the templates... in fact you can see the band size on the gel is very similar, no identical… (?) To me, this is not primer dimmer (What do you think?)… In addition, the peaks for the MC are over 80 deg, and to my knowledge you should see primer dimmers between around 70 deg. I think the big difference is in the amplification curves, where the controls are between 7- 15 CP away from my samples. Krümel, I'm sending attached a resume of my results as you suggested. I appreciate your help!
Thanks so much !!
It's your problem solved by now?! I'm using sybr green also, and sometimes i get a profile like that.. a very strange MC. I don't think that is contamination that give me a strange profile. Fortunatly, the NTCs do not have the same Tm of the samples.. so i keep using these data. It would be wonderful if my NTCs look flat on MC at all the time!!!
Krümel, about your question... YES, I have always checked the product on a gel. The melting curves for the controls have peaks in the same range as for the templates... in fact you can see the band size on the gel is very similar, no identical… (?) To me, this is not primer dimmer (What do you think?)… In addition, the peaks for the MC are over 80 deg, and to my knowledge you should see primer dimmers between around 70 deg. I think the big difference is in the amplification curves, where the controls are between 7- 15 CP away from my samples.
Guarini, the band on the gel doesn't really look like primer dimer (+Melt temp is too high), you are right. As it is not identical to the other bands, contamination is a good guess.
If you are cooking your RT-PCR long enough, something will show up. Therfore, Piko is probably right -your samples are in a good CP range, I personally would use them...
Contamination is an issue ... separate pre and post-PCR benches and pipettes, make small aliquots of everything, use barrier tips, etc. I think that there are some guidelines in several posts on this?
Hi friends, thanks for the help. As I have seen in other posts, contamination problems are very common and difficult to solve completely... With your suggestions and others PCR set-up guidelines I'm trying to solve it... Thanks a lot !