Total RNA was isolated from C. elegans, and cDNA was reverse transcribed. We can get nice PCR product whenever we amplify a housekeeping gene, or a specific one, but only short ones (maximum 800-1000 bp). When we try to amplify a longer sequence (3000-3500 bp), then it fails every time. We use the same primers, but in another combination. I controlled the primers in primer3 and NCBI primer blast as well, they are compatible with each other, and specific to the required product. I changed the primers a couple of times, tried more temperatures, and three different polymerases. There is no product. Could it happen, that the RNA is partly degraded, or fragmented, in every sample tha same way?
It is possible that the RNA is degraded, check it with a Bioanalyzer. More likely though that the "routine" cDNA synthesis will not generate 3kb templates (might run an agarose gel to check it), and try different protocols (egArticle A Highly Efficient Method for Long-Chain cDNA Synthesis Usin...
and different enzymes (https://www.thermofisher.com/us/en/home/life-science/pcr/reverse-transcription/5steps-cDNA.html)
You will also need a different PCR protocol for longer amplicons.
Dear Péter Gyarmati,
thank you for your answer. I was afraid, that the normal reverse transcription can not produce so long cDNA products... :( The PCR methods were adjusted particularly for long PCR, we can get more than 10 kb long products from other templates, so that could not be the problem... I try to check the RNA quality somehow...
hi, did you by any chance tried a Gene specific priming? In addition which RT did you use? Protocol? incubation time? There are some points to improve it just need a bit more background. I would also say 3kb is challenging but not impossible
Most reverse transcriptase are rubbish: it's an inherent limitation. They bind, they reverse transcribe a bit, they fall off. Even the best, optimised enzymes have processivities of ~1500bases.
They can bind again, and continue extension, but they can also go bind somewhere else. In a reaction primed with oligodT, sites with bound oligodT are going to be in excess of "sites already extended once" for most of the reaction, and of course, the chances of an enzyme repeatedly rebinding to the same ongoing reverse transcription get lower the more repeat events you need. Reverse transcribing more than ~5000 bases from the polyA tail is...ambitious for most commercial, standard, RT kits.
This is also the reason oligodT-primed reactions tend to be 3' biased. The further your target sequence is from the polyA tails, the less likely it is to be fairly represented in your cDNA.
If your sequence of interest lies 8,000 bases from the polyA tail, chances are it will never get reverse transcribed.
You can use random priming, which allows reverse transcription basically...everywhere: this solves the 3' bias issue, but also means your cDNA is random primed, and thus fragmented: reverse transcribed sequences can start anywhere, and (given the low processivity) also end anywhere. If your target amplicon is 3000 bases, chances are most of the sites for primer 1 are in different fragments to those for primer 2, so you get poor (or zero) amplification of your target.
There ARE reverse transcriptases that are optimised for long range: maxima H minus is one I've used:
https://www.thermofisher.com/order/catalog/product/EP0751
Here you use oligodT priming, but also leave the reaction for longer than you might otherwise (I use an hour at 55 degrees), and the processivity of the enzyme is such that while you will STILL get 3' bias, you will also transcribe all the way through to the 5' end.
For reference, I have used this to capture 5' sequence of dystrophin mRNA, which is ~14,000 bases long, so...it can manage that.
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