What is your preferred Reverse Transcriptase? - (Sep/28/2012 )
I just want to know what RT enzymes people use around the world in the lab. I will be very happy if you could share it here and explain the reason why you prefer this enzyme to other enzymes out there.
I normally use Fermentas' Premium RT, and I just recently managed to synthesize a 12 kb cDNA from a viral RNA. I think it's really amazing, although I had to optimize my PCR to get that band. I mean the percentage of the template cDNA in my PCR reaction (22%) was more than the recommended amount (10%). This way I don't have have to amplify shorter fragments and run Overlap PCR to fuse them together.
I hear that many people in the US use Superscript III. Why do you think it's better?
For common RT (human RNA for RT-PCR) we used AMV RT from... em, dunno Fermentas? But seems they even going to discontinue it.
Since we're doing real-time, we transcribe all RNA with Transcriptor First Strand cDNA Kit from Roche. Because.. it's suited for qPCR (RNAse H activity), it's a complete kit with primers and everything and we use Roche mixes and machine. Nowadays we seldom do normal RT, and if we do, we use Transcriptor as well, because we're lazy and probably we don't even have all the components for the AMV protocol anyway.
There is some Superscript in the freezer, so someone was using it, it's said that it has unprecedented yield and specificity, but I think due to reduced RNase activity it leaves RNA-cDNA duplexes, which are unprefered for qPCR.
I thought so, Fermentas and Roche must be working well in Eastern Europe. I'm just looking for an RT that can give me a long cDNA, because most RTs can only synthesize short fragments.
I used to be a Roche supplier btw
I would say the length of produced cDNA will also depend much on the primers used. Oligo dT gives you whole single mRNA strand, but if your RNA is fragmented, the percentage of 5' end will be very low (and for viral template RNA not suitable). High concentration of random hexamers will cause many transcritpion starts so high cDNA yield, but shorter products.
I think there will be different RTs ideal for different tasks. Some people want mRNA only, some people want high yield but don't care about shorter fragments, some people want linear transcription along the whole RNA population.. there probably isn't an universal one.
(Roche representatives (at least ours) are very agile in helping whenever I encounter a problem or need a solution. Also the company as whole, at least in our region is very flexible, you're choosing cycler, they will borrow you demo machine, you need this, you ask, it could be done, they could arrange. In contrast with very rigid organisation of fromer ABI, this was very refreshing, anything unusual you wanted from ABI, it was a problem, the representative was trying his best, but he couldn't do anything, we wanted to test (borrow) their new cycler, but there were some administrative troubles and everyting, they only had the older model and paperwork took months. Well, so finally we were trying LC480 and bought it. And since in our experience it gives best results with Roche mixes, we buy from them as well. ABI guy was desperately trying to sell me their mastermixes at least for several years afterwards, but when the price is similar and you get slightly better results from the company that have mixes suited for your cycler, and you have the other components working together and if anything you can ask the company what's wrong, since everything is from them.. well you see the advantage.)
I cannot use Oligo dT. My virus has a single-stranded negative-sense RNA genome. that's why it's difficult to synthesize cDNA with it. I designed several GSPs to create different sizes of cDNA. But you are right, I noticed when I use more GSP I can get sharper PCR bands.
I know the history of all those real-time PCR machines. I sold many LightCycler machines in my region when it was very popular. LightCycler actually is a patented product from Idaho Technologies, but Roche bought the right to sell it. ABI used to be popular but it's dying now. I still think the most accurate qPCR machine is LightCycler 2.0, because it was capillary based, but at the same time the capillaries were a disadvantage because of their high price. Currently Corbet's Rotorgene 6000 HRM is the best machine....although Roche's 480 is the best high-throughput among them all.