Hi,
At the moment I'm trying to acertain the endogenous gene expression of hP2Y receptor subtypes in the tsA201 cell line by reverse transcriptase PCR, however I keep having problems with bands appearing in my RT- control that we've not been able to remove despite trying multiple approaches.
Although we initially used a Quiagen RNAeasy mini kit , because of poor yields we switched to isolating the RNA from confluent cells (with a total cell count of approxmately 3 million cells) using a Bioline Isolate II RNA mini kit and following the standard instructions recommended, including doing the on-column DNAse I step (we're doubling the incubation time with the DNAse I recommended from 15 minutes to 30 minutes). We typically get fairly good yields (typically about 650ng/ul) and A260/280 and A260/230 reads using a Nanodrop 2000c and blanking with the same RNAse free water as the RNA is eluted with.
We quickly identified though following cDNA synthesis with qScript cDNA SuperMix though that the RT- control containing only the RNA seemed to be rather prominent for some receptor types (especially hP2Y1, 2 and 12). Since then we've used a post isolation DNAse I (NEB DNAse I) with heat inactivation and then going through the RNA cleanup protocol for theBioline kit, (this typically reduces the yield to ~250ng/ul) however we're still getting bands in the RT-, abit not as prominent.
In case the NEB DNAse I was being ineffective we tested doing the post DNAse I with the Bioline DNAse I included in the kit, but that seemed to work similarly. We've cut out and purified the RT+ and RT- bands and sent them away for Sanger sequencing, and in all cases the sequences correspond to the receptor being amplified, which to us suggests genomic DNA contamination.
Although I know it's generally seen as unlikely, we've also tested whether the polymerase (Promega Gotaq Green Mastermix) could potentially have RT activity by treating both the cDNA and RT- with 50 units of NEB DNAse free RNAse If for 30 minutes at 37 degrees, but comparing treated and un-treated RT- and RT+ reactions on the same gel, whilst it dramatically reduced (but didn't eliminate) the RT- band in some reactions in others it seemed ineffective or induced a double band so we decided to not use it in future.
I would just like to know if anyone has any suggestions as to anything else I could try? The water controls are clear and we're making certain to make up the positive control PCR reactions (using either plasmids or cells endogenously expressing the receptor mRNA) in a separate room with separate pipettes (we're using filtered aerosol resistant tips at all times) and reagents. Whilst we have considered doing another DNAse I we're concerned about the reduction in yield that would probably result.
Thanks!
I see that you're approaching this issue methodically and you've covered every step I would.
It seems that the DNA bands you're getting are either coming from inefficient DNAse activity or contamination in your laminar. In the first case, try getting a free DNase sample from other companies. In the other case, it's prudent to clean out the laminar and make sure that you're not carrying any contamination
Meanwhile, I'm leaving my comment here hoping someone will have better ideas.
Good luck!
Thanks for the suggestions, I was thinking of the DNAse I enzyme perhaps being ineffective and will see if I can get a sample from another company. Unfortunately I'm not working in a laminar flow cabinet as we have a lack of hoods available so I'm having to do the RNA extraction, PCR etc directly on the bench, which is why we're making so certain to prepare the positive controls in a different lab with separate resources. This project has turned into a nightmare to do as we've already had to switch the polymerase twice because one induced double bands in some samples and the other bizarrely caused bands to consistently appear in the water controls, fortunately the Gotaq Green Mastermix seems to be working well now.
Hi Samuel,
I too feel that changing the DNAse I source would be a help here. Apart form this I would suggest you give the DNAse I treatment to your purified RNA sample (and not on-column DNAse I treatment). After DNAse I treatment you can again go for second round of sample purification through column binding/elution step (though it means you will have to sacrifice an extra column here!).
Another suggestion would be to use a different source of water/elution buffer for dissolving your end product.
Hope it helps, Gud luck!
Have you ever tried polymerase with UDG? Helps with DNA that might be flying around during PCR preparation.
Also, cleaning benches before work seems to help.
To get around gDNA contamination, you can also try to design your primers to amplify ONLY from cDNA and not gDNA. It's not always possible, but in most cases have one of the primers span exon-exon junction (gDNA will have exon-intron-exon and the primer will not work there). Or design your primers in such a way that gDNA and cDNA amplifications will give different product size.
The point is, even if you can't get rid of gDNA completely, you don't have to worry about it since your cDNA bands will be different.
oh and commercial water made all the difference for me when I was doing RT-PCR.
I believe it was Sigma's RNAse free. maybe something else-free too.
Unfortunately nearly all of the hP2Y receptor genes are single exon which makes genomic DNA control very important. I've managed to get Primer Design UK to send me a sample of their Precision DNAse kit - which also usefully completely inactivates at a lower temperature compared to the NEB DNAse (55 vs 75 degrees) or so the product claims - and I should get it shortly.
I know exactly what you mean about commercial water, Quiagen RNAse free water and the RNAse free water that came with the Gotaq were working very well with no bands at all being produced in the water control for each reaction compared to previously when were having massive problems with a single band of an identical size appearing... and then the moment I started preparing the positive controls to go on the gels (in a separate room) the cursed band starts appearing again, I'm hoping it's just co-incidence...
Wait! Could it be, that your loading dye has funny stuff in it? A student of mine once mixed in DNA into stock dye, and it took time to realise what happened.
Also, if your fragment is on the exon, how do you know from sequencing, that the rt- comes from DNA?
Fortunately the Gotaq green has the dye premixed in so there can be any contamination there (somebody in the lab did suggest that). As to the sequencing results, I'm not an expert in this area (just learning!) but the person who's guiding me in this said that the results we got correspond to genomic contamination (from my understanding of what he said).
may i ask you just elute the product and do re amplification from it see if problem still remains.. if it remains u have redesign your primer
I received the Precision DNAse Kit sample from Primer Design UK yesterday (they have very efficient and helpful customer service by the way and were happy to provide samples of their products entirely free of charge) so I've got 10 DNAse reactions to use.
I'm currently extracting new RNA (from the exact same cells and passage as I was using before as I fortunately have 3 samples of the lysed cells frozen at -80) and will use the new DNAse as well as replacing all the reagents I'm using (water, primers, polymerase etc). I've cleaned the bench down with RNAse zap and have found a free hood with UV that I can use for when doing the PCR; hopefully this will pay off.
Thanks everyone for your help, I'll let you know how it goes.
I've extracted the new RNA as I said above and did the DNAse reaction with the Precision DNAse sample, but am now wondering whether I need to do a RNA cleanup on it as well? I checked around and papers which said they'd used it didn't say anything about a cleanup, but that may just have not been mentioned.
I analysed it on the Nanodrop and got a concentration of 233ng/ul, an A260/280 of 1.80 and an A260/230 of 1.57. I was a little confused as to why the 260/280 and 260/230 figures were so low (they're usually ~2.00), until the person who's guiding me in this (and is fortunately back now) pointed out that the DNAse will inherently add some protein to the sample and therefore throw the spectrophotomer readings off a bit and making them un-reliable. Therefore do you think it's worth doing a spin column cleanup? That's what we'd previously done when using the older NEB DNAse, although I suppose there will then be a loss in total yield.
I've now found that we do have a proper PCR hood in the other building so I'll be doing the rest of the PCR in that when it's available.
Yes, definitelly clean up the DNase - you don't want that in your PCR in case it survived inactivation.
Well I've cleaned it up with a spin column as I've done before, I got a fair concentration of 134ng/ul and an A260/280 ratio of 1.99, but for some reason the 260/230 ratio was still 1.57, which is surprising as usually it's above 2.00 with all the other samples I've extracted from. I've still got a 3rd sample of the same cells so I may just repeat the extraction and DNAse.
The ratio is more recommend value. The low ratio might come from additional salts in the sample. If it doesn't inhibit the pcr, I think you're good to go.
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