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In Topic: Strange RT to qPCR results with new kit
21 November 2012 - 07:45 AM
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?
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).
In Topic: Strange RT to qPCR results with new kit
21 November 2012 - 07:41 AM
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.
