5 replies to this topic

[k_undertoe]

I recently began using a different product to produce cDNA.  It is a commercial kit with a 2-step protocol, the first using a gDNA elimination step and then proceeds with an RTase from a 'new source' and also a non-specified primer mix, which is said to be good for low abundance transcripts as well as transcript from 5' ends.

SO, I have used the kit and the first time I did not generate ANY amplifiable signal even for my reference genes (GAPDH, RPS19).  I did a side-by-side prep of control fibroblast cDNA using my previous kit, so it was not an issue of bad sample.  Tech support could not come up with any reason it did not work so they sent me a new kit.  I again ran through the protocol, but this time I got signal from everything.  Genes that the fibroblasts do not express came up as positive (Oct4!).  All of my controls are clean- the RT minus control for the fibroblasts shows no amplification, my no-template water only controls come back clear too.  I did no-template controls for both my RT step AND my qPCR step so there is definitely no contamination in the mastermix.

I have no idea where this signal is coming from, considering my RT minus control is clean as well as my no template controls.  Any ideas?

[bob1]

Sounds like genomic DNA contamination.  The first run through sounds like you didn't get rid of the DNase effectively before cDNA prep or used too much RNase, which can act non-specifically on DNA sometimes.

[Trof]

Do your Oct4 primers amplify DNA at all? You can have a negative RT- but sometimes the only certainty you will have especially when trying a new kit is primers that don't amplify DNA (lying on the exon-exon border).

If not, you may also consider designing some that amplify only DNA, for testing gDNA contamination (promoter sequence or so).

[k_undertoe]

bob1, on 16 November 2012 - 11:32 PM, said:

Sounds like genomic DNA contamination.  The first run through sounds like you didn't get rid of the DNase effectively before cDNA prep or used too much RNase, which can act non-specifically on DNA sometimes.

It can't be genomic contamination because my RT minus control for the cells  came up clean.  If there was genomic contamination, that control would have had genomic DNA left in it that would have been amplified.  My first run... possibly I would say DNase could have totally annihilated my sample, I can see that may have been the issue; we don't treat with RNase so that is not it.

[k_undertoe]

k_undertoe, on 15 November 2012 - 01:39 PM, said:

I recently began using a different product to produce cDNA.  It is a commercial kit with a 2-step protocol, the first using a gDNA elimination step and then proceeds with an RTase from a 'new source' and also a non-specified primer mix, which is said to be good for low abundance transcripts as well as transcript from 5' ends.

SO, I have used the kit and the first time I did not generate ANY amplifiable signal even for my reference genes (GAPDH, RPS19).  I did a side-by-side prep of control fibroblast cDNA using my previous kit, so it was not an issue of bad sample.  Tech support could not come up with any reason it did not work so they sent me a new kit.  I again ran through the protocol, but this time I got signal from everything.  Genes that the fibroblasts do not express came up as positive (Oct4!).  All of my controls are clean- the RT minus control for the fibroblasts shows no amplification, my no-template water only controls come back clear too.  I did no-template controls for both my RT step AND my qPCR step so there is definitely no contamination in the mastermix.

I have no idea where this signal is coming from, considering my RT minus control is clean as well as my no template controls.  Any ideas?

The new kit that does not work is the QuantiTect RT kit from Qiagen, and the one we have been using that works is the Cells-to-cDNA II kit by Ambion (distrib. by Invitrogen).

[Trof]

It's still theoretically possible that RT- amplification failed/was inhibited, which made it false negative. But if you primers don't amplify DNA at all, you can be sure it's not the DNA. From that you can move to other options, sample/RNA contamination by positive sample (repeating the reaction again with new RNA will have different result), nonspecific amplification (you can sequence product to validate it's really Oct4 transcript) or simply at the end the fact, that your fibroblast actually do express Oct4 (you can try other cells which you are also sure should not express it).