How should products of first-strand cDNA synthesis look like on a gel? - (Dec/08/2008 )
Hi everyone,
I am curious to know how the products of first-strand cDNA synthesis from total RNA should *ideally* look like on a gel. I have seen many gel photos of first-strand cDNA synthesis (including my own), but the results seem to range from heavy smears without visible rRNA bands, to faint smears with bright rRNA bands.
Several posts in these forums have already touched upon this question (for example <http://www.protocol-online.org/forums/index.php?showtopic=38026> and <http://molecularbiology.forums.biotechniques.com/forums/viewtopic.php?t=9425>), but I have never been able to find a detailed description of how the gel is ideally supposed to look like. Strangely, I also don't know of any first-strand cDNA synthesis kits that provide such photos in their manuals.
1) Is the gel supposed to show a heavy smear without rRNA bands, a heavy smear with faint rRNA bands, or perhaps a faint smear with strong rRNA? (My guess is the third option. Since first-strand cDNA synthesis is less than 100% efficient, I doubt whether it can make the smear many times brighter than the mRNA in the original total RNA.)
2) Will there be a difference in result if an oligo(dT) or anchored oligo(dT) primer is used versus a random hexamer primer? (I assume that the rRNA bands will be relatively less bright when using oligo-dT primers than when using the random primers, since the latter also creates cDNA from the rRNA.)
3) If I get a smear without any rRNA bands (sometimes a rather bright smear), is this fine, or does it mean that the cDNA synthesis didn't work very well and that the smear is the result of RNA breakdown?
4) On a more in-depth technical point: can M-MuLV or AMV reverse transcriptase re-initialize cDNA synthesis many times over from the same mRNA template strand? (Since M-MuLV has a strand-displacement ability, I was wondering whether it can re-initialize cDNA more than once from the same mRNA template, using a new oligo-dT primer for each new cDNA strand. I assume that the more cDNA strands that can be synthesized from one mRNA strand during the incubation step, the brighter the resulting smear on the gel.)
Any ideas would be much appreciated!
Anton
the image will depend on the type of primer used in the synthesis: Random hexamers will copy any RNA including ribosomal RNA, while oligo dTs will only copy poly-A-tailed mRNA.
1. It depends on how much starting material you have and the primers/reverse transcriptase that you use. But basically smears are normal. Even when the rRNA is not visible.
2. I have never compared both, but I presume both would also be smears on the gel, as both would still produce varying lengths of cDNA.
3. The smear is fine. Just use the cDNA in a PCR to check, or run a spectro.
4. I would say it depends how many templates the primers stick to. The primer annealing step of 70C for 5 min prior to addition of reverse transcriptase decides how many templates have primers. Since there are no cyclic temperatures, the MMLV can only continue producing cDNAs from mRNAs (at 37C or 42C) that have primers annealed. So basically it's one RNA one cDNA.
I think most people don't show it because they don't bother to check it, and it's generally not of high scientific interest. I certainly never waste my cDNA by running it on a gel, it's just too precious. I use it in PCR directly after RT with the assumtion that RT worked, and run several internal controls to assess the quality of the cDNA. (beta actin, etc.) I never have any problems.
The intensity of the smear depends on how much you load. Try to calculate how much RNA and cDNA you would expect to have in your sample, and then use this to estimate how bright you expect your band to be. (Hint: RNA and ssDNA don't bind EtBr strongly anyway, so I would never expect a bright smear.)
Random hexamers also generate shorter cDNAs, especially if their concentration is high, so the shape of the smears might be different.
Can I ask why you are so preoccupied with verifying the products of your RT? If you are doing normal RT-PCR, don't bother running a gel; just proceed directly to PCR and do some internal PCR controls. If you are preparing a cDNA library, it would be preferable to prepare radiolabeled first-strand cDNA, run an alkaline gel alongside a labelled ladder, and then do autoradiography to determine the integrity of the cDNA alone (you won't see RNA in the radiograph).
Good question. However, I think the number of polymerase molecules in a reaction are limiting in this respect, and I think that there are a number of reasons strand displacement won't contribute to a significant increase in the number or cDNA molecules. (No time to explain, I have to go do some experiments!)
Hope this helps a bit,
Ginger
I had this exact question! The reason I'm wondering is that my PCR has failed, and I don't have a positive control to use with those primers. I'm trying to figure out if the problem is my PCR (which has been wonky lately) or a problem with my RT . I'm just not able to turn RNA into cDNA and get products, so I'm trying to troubleshoot each step.
Thanks for asking, Anton - and thanks everyone else for your help, it helped me too!