New methods to overcome gDNA contamination in RT-qPCR -

Use this category for questions related to various quantitative PCR methods, including real time PCR, reverse transcription (RT)/PCR, qPCR, etc. For general PCR methods, continue to use the DNA/General PCR forum

Moderators: r.rosati, Suzanne

New methods to overcome gDNA contamination in RT-qPCR -

Postby Sam lookingfortranscript » Dec 15 2007 7:01 pm

Hey everyone,

I am currently engaged in a project, where I want to quantify mRNA levels of several genes (some of them very low abundance) via RT-qPCR. None of the genes includes introns, so -RT (no Reverse Transcriptase) controls are essential for confirmation of validity of my experiments.

As far as I saw from reading prevoius threads, I am by far not the only person having the problem of eliminating gDNA contamination by means of DNase treatment. I extract RNA with QIAGENs RNeasy, usually digest with 3U of RQ1 DNase (Promega) in provided buffer for 1h and cleanup with RNeasy before RT with Invitrogens Superscript. For RT I used either random hexamers or GSPs.

I found previous entries very helpful and it seems to be clear (as posted before) that a 100% elimination of gDNA contamination by DNase treatment without seriously compromising RNA quality or reducing sensitivity of qPCR (e.g. less cycles) is not possible.

Because of that I was looking for alternative methods, circumventing DNase treatment. I found two articles, which appeared very interesting :

RNA template-specific polymerase chain reaction (RS-PCR): a novel strategy to reduce dramatically false positives.
http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&dopt=AbstractPlus&list_uids=1698167

Exclusive amplification of cDNA template (EXACT) RT-PCR to avoid amplifying contaminating genomic pseudogenes.
http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&dopt=AbstractPlus&list_uids=11680707

So far I didn´t get access to full text (hopefully this will change soon), but the general approach seems appealing to me. As far as I understand they use a GSP for first-strand synthesis including a 5´-tag-sequence, that is not present in the gDNA. This tag-sequence, of course, doesn´t anneal to the RNA, when rverse transcribed, but will appear in the resulting cDNA. The tag-sequence is then used as reverse primer in the subsequent PCR reaction, giving rise to a product only for amplification of cDNA.

BUT even though this doesn´t seem to be a very new idea (1990 or earlier) I haven´t seen many publications apllying this technique. Also (excuse me if I´m wrong) I didn´t find a topic/discussion of that approach in this forum, even though a lot of people appear to be struggling with gDNA problems. Since you (Suzanne and others) seem to have a great deal of experinece in the field, I would love to hear your oppinion!

There is also a freely available program for designing proper tags for this method (in perl):

http://www.ncbi.nlm.nih.gov/entrez/utils/fref.fcgi?PrId=3494&itool=AbstractPlus-nondef&uid=16820068&db=pubmed&url=http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16820068

Anyone who tried that, I would love to hear about results!

Many thanks in advance for comments and experiences with this method!
Sam lookingfortranscript
newcomer
newcomer
 
Posts: 2
Joined: Dec 15 2007 5:24 pm

More papers to check out

Postby Suzanne » Dec 20 2007 9:54 am

Hi Sam,

Interesting post and thanks for the links to those techniques. I had not heard of either before and wanted to take the time to review before posting.
Another paper to look at that might be helpful without having to re-design your whole assay is this one.

Simple enzymatic means to neutralize DNA contamination in nucleic acid amplification
John Ashkenas, James W. Dennis, and Chi Yip Ho
BioTechniques Vol. 39, No. 1: pp 69-73 (July 2005)

I think the problem often comes up where its not always possible to re-design or where a decent primer set cannot be designed to exclude DNA sequences.

In this paper, Dr. Bustin gives some guidelines about negative controls and how much distance in Ct values to have between a sample and a control:
Pitfalls of Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction
Stephen A Bustin and Tania Nolan
J. Biomol. Tech., Sep 2004; 15: 155 - 166.
http://jbt.highwire.org/cgi/reprint/15/3/155

But still, the ideal scenario is that the -RT control is completely negative. You could consider an approach such as TRIzol followed by column clean up with on-column (or off column) DNase treatment. TRIzol will get rid of the majority of DNA and the column will also remove DNA. If you include the DNase on top of both of those methods, you would really be removing the DNA with 3 different methods.
Maybe go with an OligodT primer for RT to further enrich for only mRNA.

If you try some of these newer strategies (RS-PCR and EXACT), let us know how they work for you.

Best,
Suzanne
Suzanne
ModSquad
ModSquad
 
Posts: 999
Joined: Feb 13 2003 8:59 pm
Location: Southern California

Re: New methods to overcome gDNA contamination in RT-qPCR -

Postby mchlbrmn » Dec 20 2007 2:01 pm

Sam lookingfortranscript wrote:So far I didn´t get access to full text (hopefully this will change soon), but the general approach seems appealing to me. As far as I understand they use a GSP for first-strand synthesis including a 5´-tag-sequence, that is not present in the gDNA. This tag-sequence, of course, doesn´t anneal to the RNA, when rverse transcribed, but will appear in the resulting cDNA. The tag-sequence is then used as reverse primer in the subsequent PCR reaction, giving rise to a product only for amplification of cDNA.

I don't get it (I haven't read the article, however). I don't see how a 5' tag on the GSP primer helps. When the GSP primer primes the RNA (or genomic DNA) there's the chance it won't prime specifically, or could prime DNA. Using the tag primer in subsequent PCR will amplify tha correct sequence, along with any 'mistake's the GSP primer made. I think the annealing temperature of priming of the RNA tends to be less specific and more difficult to control then annealing in PCR later on, so it seems to me that you're increasing the chance of problems by relying on the cDNA reaction priming of the RNA to give all the specificity to the PCR. As I said, I haven't read it, and may not be seeing everything clearly, but that's my first impression.
mchlbrmn
ModSquad
ModSquad
 
Posts: 3560
Joined: Dec 13 2005 1:03 pm
Location: Boston, USA

Can RT really use DNA as a template?

Postby Sam lookingfortranscript » Dec 20 2007 3:09 pm

Hey everyone,

first of all thank you very much for your comments! I was´nt aware of the paper Simple enzymatic means to neutralize DNA contamination in nucleic acid amplification, so I´ll read it as soon as I can.

Using Trizol as a first step for purification and RNeasy for cleanup seems to be worth considering as well, since people seem to have found that this works pretty well.

Now to mchlbrmn´s comment.

When the GSP primer primes the RNA (or genomic DNA)...


I was working on the assumption that Reverse Transcriptase is truly a RNA-dependent DNA Polymerase, so Primers in the first-strand synthesis should´nt prime DNA synthesis using genomic DNA as a template. Following this assumption I saw the problem of gDNA contamination at the level of PCR, where you would get product, even in the absence of any transcript, rendering even a qualitative statement about transcription (yes/no) invaluable.

If Reverse Transcriptase can use DNA as a template, you´d have the additional problem that the produced cDNA might originate from gDNA. I agree that, f that is the case, introducing a 5´-tag in a GSP would´nt really help at all.

Anyone who is sure about it, it would be important to know if I (and probably the people who published these tag-based RT-PCR methods) were working with a wrong assumption (namely: Reverse Transcriptase is an RNA-dependent DNA Polymerase).
Sam lookingfortranscript
newcomer
newcomer
 
Posts: 2
Joined: Dec 15 2007 5:24 pm

Postby mchlbrmn » Dec 20 2007 5:15 pm

Oops. I just forgot that RT should be RNA dependent. Doing a quick check with the NEB catalog wedged next to my computer, it says that their MMLV RT is an "RNA dependent DNA polymerase" that uses "either RNA... or single stranded DNA as a template". I doubt it can do much with genomic DNA. However, mayabe you could have a little contamination from carryover RT primer in the PCR reaction(?). You might check the concentration of GSP primers, and how much will be carried over. But, it's probably fine. I assume the technique is designed to avoid the genomic problem, and probably works well. I just forgot that the RT shouldn't prime genomic DNA.
mchlbrmn
ModSquad
ModSquad
 
Posts: 3560
Joined: Dec 13 2005 1:03 pm
Location: Boston, USA

RT does have some DDDP activity

Postby Suzanne » Dec 20 2007 8:32 pm

Actually, I remember reading that reverse transcriptases can have low level endogenous DNA dependant DNA Polymerase (DDDP) activity. I can't find a source or reference for that though.
I found several references saying the HIV RT has DDDP activity. But not sure about AMV and MMLV.

I thought there was an article about it on Ambion's page but can't find it now.
However, the buffer composition with the enzyme is optimized for RT activity and not DNA polymerase activity so the residual activity should be minimal.
Suzanne
ModSquad
ModSquad
 
Posts: 999
Joined: Feb 13 2003 8:59 pm
Location: Southern California


Return to Real-Time qPCR/qRT-PCR Methods

Who is online

Users browsing this forum: No registered users and 4 guests