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How to find that my RNA is not contaminated with gDNA, before running Real-Time

RNA gDNA CONTAMINATION

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#1 cancerlab

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Posted 10 November 2011 - 09:49 PM

Hi!

I extracted the RNA using QIAGEN RNeasy kit and I checked the purity (260/280, 260/230) and integrity (28s/18s) of my RNA. Both of them were perfect.

I also did not see any band near the wells or between wells and 28s while running the gel to check the integrity of my RNA samples which means no contamination with DNA or at least small contamination.

I treated my samples with Ambion DNA-free DNase treatment and removal reagents kit (AM 1906) to digest trace amount of DNA, if presents.

For RT-PCR (SuperScript™ One-Step RT-PCR with Platinum® Taq) I did not use any -RT, since I was not able to buy "Platinum® Taq DNA polymerase" itself for being used in my -RT. I got clear band with the desired size by running gel.

I have 2 questions:

1- Is it possible that the band I got in my gel (after RT-PCR) and its density be something relevant to contamination not only my interest gene?

2- To proceed to real-time PCR, do I need to check my cDNA (made by Superscript III first-strand synthesis supermix for qRT-PCR) for possible contamination with gDNA? I have heard that some people do not use minus-RT in their real time-PCR and check their cDNA instead (to prevent their real-time reagents from wasting).

It would be appreciated if you kindly advise me.

Thanks
Cancerlab

#2 Curtis

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Posted 11 November 2011 - 08:05 AM

I'm not sure if I can help you but I used a kit from Qiagen that had a gDNA removal reagent, then the kit claimed it would only amplify cDNA, not RNA. I am not sure why they claimed that, but maybe you can have a look at their protocol. I used Qiagen cDNA synthesis kit and SYBR green amplification kit. I don't remember the catalog numbers, but have a look at their website...

#3 cancerlab

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Posted 13 November 2011 - 05:39 PM

I'm not sure if I can help you but I used a kit from Qiagen that had a gDNA removal reagent, then the kit claimed it would only amplify cDNA, not RNA. I am not sure why they claimed that, but maybe you can have a look at their protocol. I used Qiagen cDNA synthesis kit and SYBR green amplification kit. I don't remember the catalog numbers, but have a look at their website...


Thanks for your reply. I'd like to know how can I find whether or not my RNA is contaminated before running real-time PCR? I know that I can use -RT in real-time, but I just want to know is there any way to check contamination before running real-time?

#4 Trof

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Posted 22 November 2011 - 03:03 AM

AFAIK you can't. You can check it on a gel, but trace amounts that can affect PCR won't show up. But you treated the RNA twice with DNAse and DNA-free kit is particulary good, we never had any detectable DNA contamination after the treatment, so I would say you probably have a contamination instead of DNA. Are those primers intron spanning? If so, the DNA product is supposed to be bigger, is it in your sample? Can you test it the

You don't need to do RT- for all samples if you are sure your DNase treatment works well, you can run like one RT- at a time for all set of samples to check it's really OK. You don't need to buy Platinum polymerase just to put it into RT-, just leave the enzymes out.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

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#5 cancerlab

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Posted 05 January 2012 - 07:14 PM

AFAIK you can't. You can check it on a gel, but trace amounts that can affect PCR won't show up. But you treated the RNA twice with DNAse and DNA-free kit is particulary good, we never had any detectable DNA contamination after the treatment, so I would say you probably have a contamination instead of DNA. Are those primers intron spanning? If so, the DNA product is supposed to be bigger, is it in your sample? Can you test it the

You don't need to do RT- for all samples if you are sure your DNase treatment works well, you can run like one RT- at a time for all set of samples to check it's really OK. You don't need to buy Platinum polymerase just to put it into RT-, just leave the enzymes out.


Thank you for your reply.
The primers used in RT-PCR are intron spanning which were ordered based on a paper and that gave me a product with the size of 450 bp. Is that ok?

Another question: Is it necessary to check concentration of total RNA after treatment with DNase? I know nanodrop can not distinguish between RNA and DNA.
I did not see any contaminated band in the gel while running it for checking the integrity. So I assumed that there was no contamination, and after Dnase treatment I did not check the concentration again.......
Have I made a mistake?

Thanks in advance




#6 Trof

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Posted 06 January 2012 - 01:09 PM

So your intron spanning primers give 450 bp for a cDNA and what bp for DNA? It should be 450 + intron. You should see this on gel or in melting profile. If you have only 450bp band then it's not DNA contamination. But it can still be contaminated with cDNA.

I think the only meaningful RNA measurement is AFTER the DNase treatment, because only then you have "pure" RNA. If there really was no DNA before treatment, the values would be the same, but then why do the treatment in a first place.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






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