I am using RNA extracted from whole adult female Brugia malayi worms for qPCR studies. I have been using iTaq Universal SYBR green RT kit for my qPCR. From the first run I am getting a separate curve for the negative controls that do not have any RNA sample. I conducted a melt curve with my first run and the negative control had a separate peak. In my second run, I ran only controls (no template). I ran the whole RT PCR run along with regular qPCR on my negative samples. Yet again, I got an amplification curve. We have two separate rooms to make master mix and to add RNA+run qPCR room. The pipettes are designated only to PCR and not used for other purposes. The tip boxes are always newly opened.
1. Have I contaminated the kit? How can I detect if I have contaminated the kit or primers with the RNA?
2. Should I have a nonRT PCR negative control in my plate to see if the amplification is due to gDNA or RNA?
3. Any other suggestion?
P.S: This is my first ever qPCR reaction. I am a newbie to this technique and I really want to learn the correct way.
You might have different answers but in every case, if you increase cycle number, often above 40-45, you will the amplification of a DNA fragment. This DNA fragment might come from different sources. It could be primer dimers. It could be very low amount gDNA. It could be some aerosol due to the manipulation various cDNA. For me RNA is not a source of contamination for PCR.
The aerosol could be avoid by the use of two places, one for the mix and one for the sample.
Yes, you could do RT neg PCR, it is a very good idea as a "real" negative control. you will see gDNA contamination. Do you do DNase I treatment before RT ?
Hi,
There are dozens of potential reasons why you're amplifying something in your negative control. Generally, I would recommend to read about contamination in "normal" PCR because everything you learn there applies to qPCR as well.
As you suggested yourself, you should routinely run a non-RT reaction alongside to check for genomic DNA contamination.
Since your melt curve in your neg. control is different to your desired amplicon (maybe run it on agarose gel), I guess it's either primer dimers (if melt temp is lower than your real product) or amplification of an unrelated sequence. You could try:
1. Raise the annealing temp during your qPCR to prevent
2. Design better primers that a) are not predicted to form secondary structures and primer dimers and b) do not misprime to other genomic target sequences
3. Reduce the number of cycles in you qPCR (as mentioned above you will almost always amplify sth if your primers are not particularly well designed)
I would quickly check the whole procedure with -RT control and from your same RNA samples using a different primer, maybe sth that is known to work in your lab. Then you'll at least know if you may just need to redesign your primer or whether you've got a more general problem (kit and lab contamination).
Good luck!
All above are well said..incase if you are planning to redesign your primers i would suggest use gene runner software for designing your primer. It will give you whole information like 1)is their any hairpin loop, @)any dimer is forming,3)what will be your primers tm, 4)what is the gc content of your primer and so on...
Good luck
I would try to use lower concentration of primer ( 5 or 2.5 pmole ) might be higher primer concentration produce more dimers
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