Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

What went wrong in my qPCR? - please look at the dissociation curve (Feb/19/2009 )

Pages: Previous 1 2 3 Next

qPCR is very, very sensitive to the concentration of primers, even if no dimers are anticipated. if you can, find ABI's User Bulletin #2 on their website. it's a pain to read, but it tells you how to do titrations of primer and template in order to get a good reaction efficiency. if you don't do this, you're not likely to get any usable results. :D

-aimikins-

molecular_medicine on Feb 24 2009, 08:44 AM said:

Hi all,

Sorry I figured out why it looked very weird.... I did not omit the empty well during analysis... now that I did it, my melting curve still looks weird, but better than before. can you please pour ur suggestions as to why it still looks weird?? please!!!!!!!!! thanks...


It's hard to tell what's what with this many lines on one graph, but it looks like each line has only one major peak, which is what you're going for. However, if all the wells are the same gene, you still have a problem, since there is a wide range of Tm's.

-gfischer-

molecular_medicine on Feb 24 2009, 08:44 AM said:

Hi all,

Sorry I figured out why it looked very weird.... I did not omit the empty well during analysis... now that I did it, my melting curve still looks weird, but better than before. can you please pour ur suggestions as to why it still looks weird?? please!!!!!!!!! thanks...


Ok, what I see when I look at this, is that you're looking at the melt curve for multiple samples right? If so, what you're seeing is all of the different melting curves, rather than an individual sample's. You need to select the wells that are for a particular primer set and look at them. Then you should see just one peak hopefully. Some of it still looks like there may be primer dimer in some samples or what not, but you have to look at the samples individually to make sense of it. Ex. (Look at one tissue, one primer set, all wells with this combo). Hope this helps.

-josurb-

I would suggest just to run the PCR machine with either no plate or a new empty plate. This will let you see if the machine is contaminated. I visited a lab who had funny plots like you. They ran it without a plate and got all sorts of amplification, so the machine had been contaminated by someone. They cleaned it and then everything was fine again.

-beth-

I used plate real-time pcr (Roche 480 --> 384 wells) and I have found that there are many conditions that can affect the specificity and efficiency of your amplification. You may need to tinker around with your experimental setup in order to optimize it. I suggest running a few test runs with different qPCR programs (ie. 2 step vs. 3 step, different annealing temperature) and also using different amounts of starting RNA. Different machines behave differently, so unless your supplier tested the primers and mastermix for the specific machine you are using, its unlikely that you will get perfect results.

If your dissociation curves aren't looking so good its probably caused by 1 or both of these reasons:
1. your sample is contaminated with proteins and other junk or is degraded, ie. your sample has low quality.
2. your primers are not specific enough and are amplifying other targets - redesign your primers or adjust your mastermix formulation, increase annealing temperature to increase stringency.

-biotechgirl-

Hello everyone, I need help with my PCR. I am new to the PCR and my aim is to detect the expression of specific genes by RT-PCR. I have enough total RNA (detected by gel electrophoresis, nanodrop) but after trancription I cant detect any cDNA. I dont know where I go wrong since I add all the reagents (random primers-70 C for 10 mins followed by 5 X buffer, 0.1M DTT, dNTPs and superscript II- 42 C for 50 mins).I would be happy if someone could help or give suggestions.

-shankares-

In my lab, we also had problem with contamination in real time PCR, by using SYBR Green in an ABI machine. We solved the problem by adjusting primer and dNTP concentrations in mastermix. These reagents in excess (i.e. in concentrations needed for conventional PCR) can cause inespecific reactions and contamination, since high sensitivity of real time PCR. I suggest also to review your primers; you should design a primer pair specifically for your SYBR reaction, for instance, by using the software Primer Express supplied by ABI. Primers for TaqMan PCR or conventional PCR not necessarily fit well for SYBR Green real time PCR. Good luck!

-jonas albarnaz-

Are you using good sterile barrier pipette tips when loading your samples? This can be the source of many of your contamination issues.

-sciencelover-

Hi

I'm working in frozen heart tissue of rat for RNA extraction. These tissue were frozen in -20 degree from 1 years ago! But I need extraction RNA from these. I tired several times by Absolutely RNA® Miniprep Kit (Stratagene) for extracting RNA. I didn't any band in electrophoresis gel, but nanodrap spectrometer was shown good ratio about 1.9 (A260/A280). so I synthesized cDNA for QPCR from that RNAs.
After QPCR I showed amplifying is good and efficiencies is good but no-RT cDNA as negative control for gDNA contamination had amplify too!
I don't know about this problem! If does gDNA exist or RNA degrade?
Thank you for your advice...

-rfardid-

You were killing RNA by 70 dC for 10 min, you can skip denaturing all together, at least denature for shorter time, say, 2 min.

-WSN-
Pages: Previous 1 2 3 Next