qPCR, no RT controls always positive (as cDNA samples) - (Jun/12/2006 )

Dear All,

I often vist http://www.protocol-online.org when i have questions or problems, since most problems are already addressed by others before. However, the current problem that i'm facing (which is i guess a common problem) is as postulated in the title, the fact that my negative RNA controls show positively in RT-PCR (using LightCycler by Roche Diagnostics).

RNA samples are extracted from a diatom (T. pseudonana) and a cyanobacteria (N. spumigena), using Qiagen's RNeasy Mini extraction kit. Elution volumes are around 30 ul, and yield (which is low in my opinio) is for the diatom (cultured cells) around 20-30 ng/ul and for the cyanobacteria even lower, around 10-20 ng/ul.

For cDNA synthesis i use SuperScriptII, with approximately 100 ng of RNA sample (Total RNA). Final amount of 20 ul is subsequently diluted to 80 ul. 5 ul is then used in a PCR reaction.

To prevent genomic DNA from contamination of my RNA i used Qiagen's RNase free DNase I On Column DNA digestion. Also, the quality of the RNA was assessed using a NanoDrop (quality seemed good, with an 260/280 of around 1.85-2.00), and on gel.

For the diatom, eukaryote, it is possible to use primers over intron/exon barriers, however for the cyanobacteria this is not an option.

So i wonder, does anyone has an idea what is going on, and even better does anyone has the solution for this. My guess is that still genomic DNA is in my RNA, (not degraded by DNase, because to dense??) but could it be that my primers are amplifying directly mRNA (i guess with Tm optimal of 60 (and PCR on this temp) that this is not possible at all.... but can't think of anything else).

Thanks in advance,

Yours truly,

Ronnie de Jonge

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Ronnie, do you use an RNAse step after your reverse transcription, it might help as Supersript II has a low RNase H activity.

I'm new to the qPCR and just asked a similar question on a seperate thread, but theoretically it shouldn't be an issue as DNA polymerase can't use the RNA as a template.

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I would still suspect DNA contamination, somewhere, being introduced into your rxns if your neg controls are showing positive results. Inefficient Dnase is usually the problem here. Do your neg no RT controls contain RNA from your minipreps?

RNeasy has an on-column dnase protocol, that you should try if you have not. It's pretty efficient and should be in the protocol that comes with the kit.

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Thanks for the responses, i've been using this RNase free Dnase I (on column by Qiagen), and it did not sufficiently affect the outcome...

I'm now trying to get rid off the dna by LiCl precipitation (7.5 M) and/or Acid Phenol-Chloroform (5:1 pH=4.7)

Hope that this works...

oh and yes all samples contain visible RNA on 2% agarose gel; and no visible DNA is present (by eye)

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Is it not possible to purchase mastermix with the UNG enzyme thereby removing the possibility of RNA contamination?

I found with some of these kits to remove genomic DNA they did not work very well. Best to stick to the old fashioned way of using a DNase step and checking by gel afterwards than using a kit that is inefficient

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