[bagnall.lab]

Hi Wishingfly,
Do you have a bp size or identity for the large peak in your brain SSII +RNA sample?
It seems much like one of the peaks in our sample that pops out around 937-940bp. (It is the last of the odd peaks from post #92 that I haven't been able to eliminate yet).
Thanks

[bplevi]

Which is the lot number of the "bad" SSRT II? We are running out of the old one and now I´m a bit concerned...

btw, Protoscript II worked ok in my hands (not impressed, though), but Superscript II was clearly better. As alternative I would suggest to use PrimeScript from Clontech, also a MMLV-based RT.

Our old SuperScript II lot that worked very well was: 1316052. We found the following lots of SuperScriptII have problems: 1685466, 1706961, 1674426 and 1685468.

We tried Superscript III, SuperScriptIV, and Protoscript2. Protoscript2 gave an amplification curve that most closely resembled SuperScript2, but we are only now sequencing those samples. Superscript III consistently amplified but gave a large background peak. I’m not comfortable commenting much about the performance of SuperScriptIV since we’ve only tried it a couple times and got different results.

Simone, could you provide any more details on why you weren’t impressed by Protoscript2, and why you would prefer PrimeScript? Thanks

[wishingfly]

Our old SuperScript II lot that worked very well was: 1316052. We found the following lots of SuperScriptII have problems: 1685466, 1706961, 1674426 and 1685468.

We tried Superscript III, SuperScriptIV, and Protoscript2. Protoscript2 gave an amplification curve that most closely resembled SuperScript2, but we are only now sequencing those samples. Superscript III consistently amplified but gave a large background peak. I’m not comfortable commenting much about the performance of SuperScriptIV since we’ve only tried it a couple times and got different results.

Simone, could you provide any more details on why you weren’t impressed by Protoscript2, and why you would prefer PrimeScript? Thanks

On our hand the bad SS II RT is #1685466, which is in your list as well; the good old one is 1415529. Thanks for your first hand experience with ProtoScript II; I am thinking about try both.

[wishingfly]

Sorry I cannot access to the bioanalyzer (and its operation software) at the moment, but I will check it out the next time I use it, and keep you updated soon. But it seems to me our results are comparable as to the contamination part; would you mind posting your SS II RT lot#? Do you have any comment on using SS IV? You said you tried it out. Thanks a lot!

Hi Wishingfly,
Do you have a bp size or identity for the large peak in your brain SSII +RNA sample?
It seems much like one of the peaks in our sample that pops out around 937-940bp. (It is the last of the odd peaks from post #92 that I haven't been able to eliminate yet).
Thanks

[Simone78]

Simone, could you provide any more details on why you weren’t impressed by Protoscript2, and why you would prefer PrimeScript? Thanks

For the Smart-seq2 paper I tried different RTs and Protoscript was one of them (although it is not included in the Suppl Info because the paper was already in press). I never sequenced anything but from a test both with tot RNA and cells I always got a lower yield and lower average size. that´s why I didn´t continue further with it. I should have the Bioanalyzer plots somewhere, I can take a look.
I just got a free sample of Primescript and from a preliminary test it worked very well, maybe even better than Superacript II. Unfortunately it was difficult to get it from Takara (production problems?) and after waiting for a couple of months (!) we canceled the order and never thought about it again.
However, PrimeScript is used in several papers and seems to work very well. If I remember correctly, it is the enzyme that the group of Piero Carninci (CAGE protocol, among others) is using in all its papers.

[bagnall.lab]

Hi Wishingfly and Simone,
Our SSII lot was 1674426 but has yielded nothing spectacular , or anything other than junk, for us but we don't have any older lot for comparison. We do get better results using the SSIV, but haven't tried other options yet.
Cheers

[bplevi]

Hi Wishingfly and Simone,
Our SSII lot was 1674426 but has yielded nothing spectacular , or anything other than junk, for us but we don't have any older lot for comparison. We do get better results using the SSIV, but haven't tried other options yet.
Cheers

Hi bagnall.lab,

That lot is one of the lots that failed for us as well (reported above).

[Simone78]

We have only lot # 1660818 in the freezer (expire July 2016) and it has worked very well so far.

[longwood]

Thanks everyone for sharing your SSII troubles.

I also got bad results with a recently acquired batch of Superscript II; an older batch was working fine. Good: 1631423; Bad: 1685467.

[wishingfly]

For the Smart-seq2 paper I tried different RTs and Protoscript was one of them (although it is not included in the Suppl Info because the paper was already in press). I never sequenced anything but from a test both with tot RNA and cells I always got a lower yield and lower average size. that´s why I didn´t continue further with it. I should have the Bioanalyzer plots somewhere, I can take a look.
I just got a free sample of Primescript and from a preliminary test it worked very well, maybe even better than Superacript II. Unfortunately it was difficult to get it from Takara (production problems?) and after waiting for a couple of months (!) we canceled the order and never thought about it again.
However, PrimeScript is used in several papers and seems to work very well. If I remember correctly, it is the enzyme that the group of Piero Carninci (CAGE protocol, among others) is using in all its papers.

Hi Simone, are you talking about this product? I called the Clontech, and it seems to be readily available, perhaps the retailing is different in Europe. I just want to double check with you that we ordered the right thing.

http://www.clontech.com/US/Products/Real-Time_qPCR_and_Reverse_Transcription/MMLV_RT/cDNA_Synthesis/PrimeScript_RT?sitex=10020:22372:US

By the way, when using PrimeScript in RT, is there any difference of the protocol, like incubation time? I noticed that in the PrimeScript official manual, it says 65C, 5 min before RT; and 42C 30-60 min for RT. Do you follow this in the SMART-Seq2, or use exactly the same condition as SuperScript II? Thanks a lot!

[Simone78]

Hi Simone, are you talking about this product? I called the Clontech, and it seems to be readily available, perhaps the retailing is different in Europe. I just want to double check with you that we ordered the right thing.

http://www.clontech.com/US/Products/Real-Time_qPCR_and_Reverse_Transcription/MMLV_RT/cDNA_Synthesis/PrimeScript_RT?sitex=10020:22372:US

By the way, when using PrimeScript in RT, is there any difference of the protocol, like incubation time? I noticed that in the PrimeScript official manual, it says 65C, 5 min before RT; and 42C 30-60 min for RT. Do you follow this in the SMART-Seq2, or use exactly the same condition as SuperScript II? Thanks a lot!

It is exactly the product you posted. It was some time ago when I tried it, maybe now in Europe they have better retailer and it is easier to get it.

Regarding the annealing, I actually don´t take extra care about denaturation. I just did the denaturation at 72 degrees like I always do. I think the 65 degrees is more important when you use random primers, because they might fall off at higher temperatures. I think that 72 is better for unfolding the RNA and allow better priming of the oligo dT. Of course higher temperatures damage the RNA to a larger extent than lower temperatures but 2-3 minutes shouldn´t matter so much...at least this is always what I hope every time I start the protocol I then performed the RT for 90 minutes, just to be extra sure I got every single transcript but I probably overkilled it (the enzyme is probably already dead).

[snotarba]

Has anyone tested the Maxima RT from LifeTechnologies? Do you know if it performs well in the SMART-Seq2 protocol?
Thanks a lot!

[Simone78]

I used the Maxima H- but honestly I don´t remember the details. I think it worked, maybe less good than Superscript II. You can try to figure it out from the Suppl Table 1 of the Smart-seq2 paper, where I tested a bunch of different conditions (if you look for "Maxima H-" you will find a few experiments). Experiments were done some time ago and, to eliminate the cell-to-cell variability, I was using 1 ng tot RNA for testing different conditions (no single cells).
/Simone

[snotarba]

Dear Simone,

thanks a lot. If I've got it right from your Table the Maxima RT gives a similar lenght of cDNA on average, but lower and variable yield.

[Kneu]

It is exactly the product you posted. It was some time ago when I tried it, maybe now in Europe they have better retailer and it is easier to get it.

Regarding the annealing, I actually don´t take extra care about denaturation. I just did the denaturation at 72 degrees like I always do. I think the 65 degrees is more important when you use random primers, because they might fall off at higher temperatures. I think that 72 is better for unfolding the RNA and allow better priming of the oligo dT. Of course higher temperatures damage the RNA to a larger extent than lower temperatures but 2-3 minutes shouldn´t matter so much...at least this is always what I hope every time I start the protocol I then performed the RT for 90 minutes, just to be extra sure I got every single transcript but I probably overkilled it (the enzyme is probably already dead).

Simone,
First of all, thank you for having such an active role responding to questions on this forum, it is really great! Second, based on all the posted issues with superscript II and your comments on primescript, I am also interested in changing to this enzyme (Thank you wishingfly for posting the link). In your post above you said "performed the RT for 90 mins", does that mean that you did not include the PCR steps at 50 degrees when using Primescript? So instead you would perform the oligo dt annealing rxn at 72, assemble the RT reaction with all the same components except the new enzyme and buffer, and run a 42 degree PCR for 90 mins followed by 15min at 70 degrees. Does that sound correct?
Thank you in advance for all your help!