Q-PCR questions: Primer design and melting curve - (Apr/06/2006 )

Hello,

I am new to Real time PCR. I have a lot is questions is regards to the same. Any help and suggestions are appreciated.

1. We use Bio-rad mini opticon in our lab. Does anyone use this machine.

2. I am using TNFa and IL-1 as target genes. I am getting a signal around the same cycle for no template control as I am getting for my target gene. The message level is around 25 cycles even with undiluted cDNA. With diluted its even later.

3. With TNFa and IL-1 primers with No amplication control I am getting a signal as well.

3. How do I do a melting curve for my no template and no amplifcation control on this machnine. Do I run the regular RT-PCR and add a melting curve step.

4. For primer design I am using primer 3 program. has anyone used the Primer quest program on IDT (Integrated DNA technology) website. I think I need to redesign primers.

Is anyone using TNFa and IL-1 as target, please reply ASAP. I need help because I am really frustrated with the problems I am having. Thanks

All right, first slow down and relax, OK? remember your Einstein... "if we knew what was going to happen, it would not be called research, now would it?"

if you do a search for a member named Soluene and dig through her old posts I suspect this well help you; she came on several months ago when she was new to qPCR and she has posted many questions that probably parallel what you need to know.

I suspect maybe your hunch is right, though...could be a problem with your primers. That's frequently the hard part when you're getting set up. There is also some chance that it is contamination of your reactions...have you done any sort of experiment to rule this out? the fact that your no-RT control gives you a nice Ct is not so good. Have you run a gel of your products? This may be able to determine if it's primer-dimer, or perhaps to rule it out (and, yeah, melting curves are a good idea...I don't know your machine, but the instructions should be in the manual?). Have you tried a straight PCR with your primers, and run a gel? This can tell you a great deal about what you're seeing..as far as whether there's contamination in your cDNA. You can also run straight PCR with various reaction components if contamination turns out to be the problem, in order to find the bad egg in your fridge.

what other controls have you done? what have you done to optimize your primer and template concentrations?

well, hopefully we can help get you sorted out!

Thanks for the reply. I have not run a gel yet. Thats a good suggestion. Right now I am trying to run a melting curve on the machine to check for primer dimers. If there are primer dimers then I will design new primers for TNFa and IL-1. Plus I will change water and do the RT-PCR reaction again and see what happens. For GAPDH the no template did not give a signal. It remained below the threshold line. The no amplification control gave a signal but at 35+ cycles which I read in the literature can be neglected. Hence I think the problem might be with the TNF and IL-1 primers. Did you get a chance to see the attachment. I tried to look for a member by the name of Soulene, but could not find such a member with QPCR problems.

All right, first slow down and relax, OK? remember your Einstein... "if we knew what was going to happen, it would not be called research, now would it?"

if you do a search for a member named Soluene and dig through her old posts I suspect this well help you; she came on several months ago when she was new to qPCR and she has posted many questions that probably parallel what you need to know.

I suspect maybe your hunch is right, though...could be a problem with your primers. That's frequently the hard part when you're getting set up. There is also some chance that it is contamination of your reactions...have you done any sort of experiment to rule this out? the fact that your no-RT control gives you a nice Ct is not so good. Have you run a gel of your products? This may be able to determine if it's primer-dimer, or perhaps to rule it out (and, yeah, melting curves are a good idea...I don't know your machine, but the instructions should be in the manual?). Have you tried a straight PCR with your primers, and run a gel? This can tell you a great deal about what you're seeing..as far as whether there's contamination in your cDNA. You can also run straight PCR with various reaction components if contamination turns out to be the problem, in order to find the bad egg in your fridge.

what other controls have you done? what have you done to optimize your primer and template concentrations?

well, hopefully we can help get you sorted out!

that's because it's spelled like this:

SOLUENE paste it, perhaps? I mean, there were a LOT of good posts that can probably help you

I looked at your pix, but without anything to compare them to they are not helpful. they just show that something is being amplified

melting curve aside, please do run a gel...better safe than sorry at this point...optimization sucks. it's tedious and frustrating, and necessary not to skip anything that might be important.

again, what have you done to optimize primer and template concentration?

Can you tell me what % agarose gel I should run and which buffer you recommend making and running it in. Should I load my unknown, -template, -RT as well as standard on it. Thanks.

absolutely.

run all your samples

particularly if it's an issue with primers, or if you have a contaminated reagent

the gel...that's up to you. my products for qPCR are all about in the 90-105 bp range; when I was optimimizing and needed to run a gel, I typically ran a 1.3%-1.5% agarose/TAE gel, and because they're tiny and it's hard to see small differences, I would typically run it a little more slowly to get maximal resolution. but this is about preference and what materials you have on hand; just run a gel in your usual way if you want to differentiate between small fragments

This might be a silly questions. But do I have to run the product I prepare for RT-PCR on the gel. Also if there is some issue with the primers, what do I expect to see on the gel.

'product I prepare for RT-PCR'?? do you mean, the reaction components, prior to amplification? I do not think you need to run these

if you have a problem with your primers, it is most likely dimers or mis-priming...both of which you will see on the gel, do you see what I'm saying?

OK, so you mean I should run the primer on the gel. But I am getting a signal with no template and no RT as well. That may not be necessarily due to mis-priming. It can be due to some other source of contamination right. Please advise.

OK. I think it would be good to run the gel and post a pic

I have also asked a couple times, and this is important: what have you done to optimize primer and template amount that you are adding?

if you are getting a signal with no template and no RT controls, then I think primer-dimer needs to be ruled out. Have you read my posts?

did you run a melting curve? if so, what was the result?

and yeah, there could be some other contamination. I would also recommend running a straight PCR with various components to see if you can isolate a source of contamination