qRT-PCR no of cycles?? - (Oct/21/2009 )

Hey,

I am new to qPCR and hve a question or two - if anyone had any help with them - it would be great!!

I have some gDNA contamination - but I have already DNAsed my RNA twice (once in the RNA extraction kit, and once after the extraction). But when I did my RT negative PCR using primers and probe which dont span an intron I was getting a signal - thing is the Cp value was the same for all of them at 35.

Does this mean I can go ahead as the level of gDNA is the same in all of them, negating any effect on my results when I use my cDNA samples? Also do you have to use a 40 cycle run, if I did a 30 cycle run would the contamination not affect the results?? Or does the cDNA need to be 100% clear of gDNA?? Any advice anyone?

Also just wondering I was going to run three separatesample preps, does each prep need to also be in triplicate?

Many thanks for any help!!

scistudent on Oct 21 2009, 09:18 PM said:

Hi,
seems to me you have a multi-step setting plan.
first of all make sure your DNAse works. having gDNA after DNAse (twice!) suggests either a ton of gDNA or a problem with DNAse. check it.
also check your primers and probe. it's not clear to me what do u mean when u say "primers and probe which dont span an intron". are your primers and probe on the same exon? if yes try another set of primers on different exons that should make it. Personally i don't like chosing my exp setting to make sure that problems do not show themselves. do your 40 cycles qPCR clear of gDNA or change primers. your reasoning on high cycles not affecting your results may be sound today with this exp setting but could not be working tomorrow. better spend some time solving a problem today than a lot of time trying to understand IF there's a problem tomorrow. we're in a world FULL of known and unknown variables, try not to add avoidable ones.
that's just a personal advice.
bye
Fizban

Hi,

Thanks for the reply,

The first DNase step was during my RNA extraction - and when I found gDNA contamination I bought new DNAse to try to eliminate the problem. The product data says doing a DNAse step twice (with the new DNAse) is not advised, and I followed the protocol exactly so I am not sure why I still have some gDNA present...

The qPCR system we use has a website which tells you what primers/probe combination to use so changing my primers is not a possability as for my gene there is only one primer/probe option. Sorry if I was unclear - what I meant was that there is no intron within the sequence I am amplifying, so can't rule out gDNA amplification, all other targets have an intron so no gDNA amplification can occur.

Should I just keep re-DNAsing the RNA until absolute 0 gDNA?

i don know if this helps but as i understand... if your pcr reaction is 40 cycles and your Cp value is 35, that means there is very very residual contamination of g DNA (next to nil). So you actually need not worry at al!!!
You can confirm this by adding a known amount of gDNA and relating with the Cp value as you know 10 times increase will lead to a change in 3.3 cp value...

Pradeep Iyer on Oct 23 2009, 04:29 AM said:

Oh really, thats great news! Thanks! I'm sure this is a silly question, but is it 10 times increased after 3.3 cp due to the exponential nature of each cycle.. eg. 2-4-16 in three cycles?
I'm still a total novice at this but reading to try get to grips with it, theres just a lot out there to read!

Ideally the product doubles every cycle, so it goes 1, 2, 4, 8, 16, 32....
The number of cycles needed to amplify by 10x is log base 2 of 10, which is approximately 3.3

ya phage434.. that is exactly ther eason..
so scistudent... i think you have nothing much to worry.. as tehre is no contamination in your product...
BEst luck!!!
Do write in if you observe something else..