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-   -   Troubleshooting for real-time RT-PCR (http://www.molecularstation.com/forum/pcr-polymerase-chain-reaction-forum/87739-troubleshooting-real-time-rt-pcr.html)

luckyjihl 03-22-2013 02:13 PM

Troubleshooting for real-time RT-PCR
 
Hello all,

I have a problem regarding my recent real-time RT PCR experiment. I am studying gene expression of Bax and Bcl-2 with the endogenous control of GAPDH. The problem is that I always have an amplification in my no template control and the pictures below tell all:

http://imageshack.us/a/img803/7904/b...ionplotedi.jpg

http://imageshack.us/a/img694/7607/b...urveedited.jpg

http://imageshack.us/a/img208/6105/b...tionploted.jpg

http://imageshack.us/a/img526/3858/b...urveedited.jpg

http://imageshack.us/a/img823/631/ga...ationplote.jpg

http://imageshack.us/a/img259/6199/g...urveedited.jpg


Note: I use 100 nM for both forward and reverse primer of Bax gene and 200 nM for both Bcl-2 and GAPDH. Many probably will ask me to consider of designing new set of primer but as I am an undergraduate student and this study is my final year project. I am in short of time and money. Ordering new set of primer might need a long period of time and I just cannot wait any longer. The primer set by the way for each gene is referred from published journals. I hope someone can provide an answer to my problem. Thanks in advance.

luisillo 03-22-2013 03:59 PM

Re: Troubleshooting for real-time RT-PCR
 
Have you done these experiments using conventional PCR (qualitative) and running the product in agarose? Try it. If you still getting positive results (bands) in your no template control, it means one (or more) of your reagents is contaminated.

You should check your PCR mix reagents one by one. You can do it in the same run, just run few reactions, for instance four tubes for reagent tested. I highly suggest you use two negative (no template) controls in each test: you must work with one BEFORE you handle any DNA (cDNA in your case), this one will let you know if the contamination comes from your reagents; the second neg. control must be worked after you work your samples (with cDNA), this one will let you know if the contamination is due to handle issues.

I advise you, these might take you some time but if done it right it will save you plenty of reagents (= money). Remember to do these test using conventional PCR and agarose gels.

Good luck.

luckyjihl 03-23-2013 03:32 PM

Re: Troubleshooting for real-time RT-PCR
 
Thanks!!!
I really appreciate your kind response. but I would like to discuss with your further sir. I actually mixed the reagents and primer in one PCR tube before it was being distributed to 4 different reaction tubes (2 target gene samples and 2 no template controls). After that cDNA and water was added separately. The water is referring to the portion supposed to be taken up by template (cDNA). Let's just say we have a contamination in reagents or primer's stock or whatsoever. The primer is not so specific. It would bind to other cDNA (not the target gene) or gDNA. If that is the case, amplification of non-specific product would occur in every tubes (4 of them). However, somehow non-specific product only appeared in no template control tubes in all genes except GAPDH proven by the melt curve provided above. And if product is generated from gDNA, logically the product would have a higher melting temperature. Is my analysis correct?

Anyway, I would try out your suggestion. Thanks again for the help.

butters 03-25-2013 02:34 AM

Re: Troubleshooting for real-time RT-PCR
 
What luisillo said is correct to check for reagent problem.

by the way can you give few detail
1. Are you using home made real time mix or commercial mix?
2. Do you follow specifically the one on published paper? Regarding the amount loaded and such. Check up with your supervisor to contact the author AFTER you confirm no problem with your reagent.

From the look of your layout, you are using single tube format and not strips. Are these tube sterile?

I believe you kept your reaction? If yes do run them on gel now and load more volume then usual.

Update us.

luckyjihl 03-25-2013 11:11 AM

Re: Troubleshooting for real-time RT-PCR
 
I have posted a reply to luisillo, it said moderator's approval is required.

luckyjihl 03-25-2013 11:33 AM

Re: Troubleshooting for real-time RT-PCR
 
I have posted a reply to luisillo, it said moderator's approval is required.

Anyway I think I will write again, what I was trying to say was that I was confused with the result as if really contamination happened, both targeted gene sample and its respective no template control would have same two peaks in the melt curve analysis if I mixed the reagents together (1 tube being transferred equally to 4 tubes, cDNA added separately), am I right to say that? Now the no template control had a distinct product judged from the melt curve performed (for Bax and Bcl-2) except for GAPDH which clearly has an amplification exactly the same as the targeted gene sample. Is that really a sign of contamination?

Anyhow, I would check the real-time PCR product I had on agarose gel.

For luisillo: I don't quite get you about the reagent testing. Do you mean testing my real-time PCR mix by running it on conventional PCR or testing my real-time PCR mix by running real-time PCR and afterwards running gel to see the bands?

For butters: the mix is purchased commercially, it is sensifast SYBR hi-rox kit from bioline. From what I mentioned that I followed the published paper was the primer sequence. And I tried the cycling method also, but it did not work. Everytime I run the experiment, at least 2 genes' NTC would have amplification.

I am actually using strips, just that in order to avoid contamination, I cut the strips into 4-tubes strips. Once I finished mixing for one gene sample, I would straightaway close the tube with the lid. The 8-tube strips are supposed to be sterile I guess??

Do correct me if I said or did something wrong. Pardon me as this is my first time doing real-time PCR. Thanks.

luisillo 03-25-2013 04:40 PM

Re: Troubleshooting for real-time RT-PCR
 
I don't think you could try the testing when using a commercial PCR mix. Besides, since it's a real time PCR mix (it is, right?) there's no point in running a gel, since that suggestion was in order to you avoid wasting probes.

In your case I believe you should try another PCR mix, since is your most probable source of contamination. Also, be careful to mantain sterile your disposables (tubes, stripe tubes, pipette tips), area of work, and pipettes; so to avoid further trouble.

Remember: your negative controls (no template) should not show any amplification and all your samples with template should at least give signal to GAPDH, including positive controls and samples to analyze.

Keep it up and good luck.

luckyjihl 03-26-2013 12:05 PM

Re: Troubleshooting for real-time RT-PCR
 
I will see whether the contamination band really exist through gel electrophoresis. I will update tomorrow about my finding. Only one each though will be picked for gel running, so there are total 6 which comprises of 1 NTC for each gene and 1 targeted gene sample for each gene.

luckyjihl 04-07-2013 10:06 AM

Re: Troubleshooting for real-time RT-PCR
 
sorry for late update..
From the gel, I cannot see clearly what was inside Bax and Bcl-2 but for GAPDH, there is a clear low bp band which I suspect as primer dimer. But this confused me more as the melt curve showed that there was an amplification same with the wanted product instead of primer dimer. Any idea what is happening?


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