We are working with SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara). When we prepared libraries for the first time, results was acceptable – we get libraries from control RNA (total input amount 0,25 ng/μl) and from LCM tissue (track A-C in attached file) with one exception – low yield (approx. 0,8 ng/μl in all samples). However, all our next attempts to create libraries were unsuccessful and we cannot find the reason. We took into account all recommendations about sample storage, preventing contamination and all general requirements. Before work with RNA we treated workflow with RNaseZap, before work with cDNA – DNAZap. Kit didn’t work even when we used only control RNA with different input amount (0,75 ng/μl, 10 ng/μl and even 20 ng/μl). In attached file, I demonstrate TapeStation electrophoresis from all stages of our experiment. Seems to be, problems appear in the beginning of protocol, after stage of first-strand cDNA synthesis and PCR1 (where is no necessary amplified products on the electrophoresis).
Is anybody facing with the same problem?
Many thanks
Tatiana,
I agree that from your gel image it appears you are losing most (if not all) of your material in the first cDNA synthesis step.
It could something from the RNA extraction. or something in this step. If the image above is from control RNA, then it has to be the synthesis step.
Try contacting your Clontech / Takara rep. In my experience they are very responsive & helpful in troubleshooting their protocols.
Best of luck!
Selene.
Xin, Sélène,
Thanks for answers!
In order to understand why the kit does not work, we used only control RNA, therefore, we can exclude errors in RNA extraction or wrong time of heating (because in case of control RNA fragmentation time is determined by recommendation in user manual).
We already connect with Clontech laboratories (Takara) and told them about our problem. Unfortunately, they refused to help us, explaining that we bought a kit not from the official distributor Takara in Russia, but with the help of our foreign colleagues (price for a kit in official Takara distributor in Russia is twice as high, compared to Takara official).
We have generated cDNA libraries with new SMARTer Stranded Total RNA-Seq Kit v2, the result is very good (pic. below). Therefore, we can conclude that the first kit does not work. Can you guess, what was destroyed? It is interesting, because all two kits were recently bought and arrived in the same package. Moreover, first kit, when we prepared libraries for the first time, was in working order.
Hi Tatiana,
sounds like the enzymes from your 1st kit got left at room temp after your first prep? Sorry to hear about price differences, and that Takara wasn't more helpful, but glad you figured it out.
To be safe in the future, my standard SOP for kits is as soon as they arrive I keep them as close to shipping temp as possible, so if they arrive on dry ice store at -80, -40 or whatever your lowest temp freezer is until ready to use. After 1st thaw, keep everything on ice whenever in use, store kits at -20 between uses, and have a thermal insulated "enzyme box" in your freezer just for enzymes. Only remove enzymes from the enzyme box log enough to add them to appropriate master mix, then return immediately to enzyme box! This will guard precious enzymes against temp fluctuations or possible freezer malfunctions and ensure maximum life & effectiveness.
Best of luck!
Selene.
We have had the same problem....we have not used the stranded kit as yet- but thanks for this discussion.
Thank you, Kartiki!
We have already solved this problem, it was troubles with indexes. Now we have other problems, it is about negative control. When we prepare libraries after RNA extraction, in negative control always synthesized a libraries with low concentration, near 500pg/mcl or less. At the same in simple negative control without RNA extraction there are no libraries.
Oh NO! I just heard that TAKARA has taken over Rubicon and now I am not getting the library prep kits. What was the problem with the indices? In fact we got amplified cDNA but poof no library synthesis. I did not think the indices may be an issue.
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