Self priming in RT ?

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Self priming in RT ?

Postby bcollet » Nov 26 2004 3:58 pm

Hi There,

I recently made some cDNA from total RNA using oligo dT. I amplified by real time PCR the ELF (elongation factor) gene (taqman probe in a splicing site). I had amplification in a no-oligo-dT control.

The difference in Ct between +oligo-dT and - oligo-dT was about 1.5: not so high !

Have you ever noticed this ? Is this the result of some kind of self priming of RNA ?

I was using Invitrogen MMLV RNseH minus enzyme at 37 degrees for 1h. Would a RT working at higher temperature prevent self priming ?

I would be grateful for any information that would help understand these results.

Thanks in advance,

Bertrand Collet
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Postby John Buckels » Nov 29 2004 9:44 am

It sounds more likely to be due to gDNA contamination. Did you DNase I treat your samples? As a check, you could try amplifying an intronic amplicon from your no RT control
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Postby talbot3 » Nov 29 2004 3:00 pm

This is a common problem. Esp in DNAase-treated samples. The small oligos left over from DNAase treatment will prime the RT step-sometimes pretty efficiently. This creates a problem with consistency of RT steps. The solution is to repurify the RNA (on a column or by alcohol ppt) after digestion.

You can also get aggressive in your primer design with respect to intron-exon boundaries--then eliminate the DNAase treatment step.

Good luck

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Postby marksilby » Nov 29 2004 5:21 pm

I get this problem in no-primer controls with prokaryotic RNA in RT-PCR sometimes. The degree to which the problem occurs is variable between samples, and in all my messing around I have found no real pattern as to when it occurs. I think that the small DNA fragments produced by the DNaseI treatment are a problem, but are not the complete story. Even when I go through a second column purification after the DNase, I can see this artefact. So probably there are some fragments of RNA that are coming through and priming the reaction. The only solution I have come up with is to test each RNA prep when I make it, before embaring on a big experiment. If the artefact is there, I discard and start again. Not the best solution, I know, and I would love to find a better one!

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Postby bcollet » Nov 30 2004 8:11 am

Thanks for your replies. I am confident it is not genomic DNA contamination as all my Taqman probes are designed within a intron-exon boundary and have been checked previously not to amplify pure genomic DNA but only cDNA.

My other question would be: If this self-priming occurs, is this a problem for gene expression studies ? Assuming everything is standardised using a good housekeeping gene ?

Bertrand
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Postby talbot3 » Nov 30 2004 3:20 pm

It is not necessarily a serious problem if you have artifactual random
priming in your RT reactions. You might have a problem with consistency in the RT, because variable amounts of this DNA junk will be present from RT to RT. Since this stuff will allow RT of rRNA, you will have this to deal with. The problem would show up as difficulty in getting replicate RT-PCRs to behave, ie the Ct values would be hard to reproduce.

In the case of prokaryotic RT-PCRs, I have heard that some Taq preps have contaminating E coli DNA in them.

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Re: Self priming in RT ?

Postby Ms_Cuervito » Aug 09 2018 7:50 pm

Hi, actually i have the same problem with my RNA control without gene especific primer. In PCR normal (no Real time), when i run the control sample in agarose gel i can find some products. The selfpriming is real or not? My boss is thinking that probably it´s me who contaminate the sample... Any recomendation?
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Re: Self priming in RT ?

Postby 29yrsExperience » Aug 10 2018 3:21 pm

I did some "reading up" on the enzyme type you are using for RT, and at least in one catalog (NEB) it says that doing the rxn at 50 degrees C provides "higher specificity, higher yield of cDNA, and more full length cDNA product." So maybe that would help. That is assuming the supplier of your enzyme also claims it has higher thermostability. I have most experience with Invitrogen Superscript III, and I recall that worked at 50 C.

It sounds like you designed your own assay rather than buy a pre-made TaqMan assay. Did you do Primer-BLAST on your primers and probes to make sure they are unique for your target? Are there any psuedogenes? Maybe you need to sequence some of the products you are getting in your control reactions to find out what they are.

Good luck!
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Re: Self priming in RT ?

Postby Ms_Cuervito » Aug 10 2018 5:56 pm

Thank´s for your answer, i use MMLV retrotranscriptase from Invitrogen (works at 37 °C), and yes i designed my gene specific primer (GSP) using Vector NTI software and then run the simulation in Primer blast, i made a 28 nucleotides reverse primer and i ran PCR control using this primer to see specificity and i can see the predicted PCR product. But in my control reaction (+RNA, -GSP, +dNTPs, +RT (and 5x, dtt and RNAse inhibitor) i can find some genes and my interest gene yet withou GSP or random hexamers in RT reaction.
Maybe you are right and the low temperature in RT reaction is the problem. :idea:
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