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Old 05-04-2011, 06:41 AM   #1
joewales
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Default cDNA library quality problem

Hi everyone,
We have made a cDNA library using the Roche rapid library prep kit, using 200ng starting mRNA (we have very little starting material). The fluorometer reading of the library says that the concentration is within the desired range - well above the minimum. However, when we test it on the bioanalyser to check the size range we don't get anything - just a blank electropherogram. I tested the library with a PCR for 2 genes we are interested in and they were both positive so there must be something there.
Has this happened to anyone else? Does anyone have any suggestions on how to overcome this problem?
Thanks,
Joe
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Old 05-10-2011, 05:39 PM   #2
grassgirl
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Hi Joe,

Yes, I had this problem as well. And I have had more problems with the Roche cDNA library prep. One problem that I overcame was optimizing the fragmentation. I needed to increase the time of the incubation step. This gave better fragmentation of the mRNA going into the cDNA prep. Now the problem is getting enough cDNA and library material to proceed to the emPCR step. I don't know if a lot of the ds cDNA is being lost in the AmPure Bead purification step, but that is my guess.

I did run a substandard library after 2 rounds of emPCR and got a fraction of the reads that one is supposed to get. I got around 15K reads instead of 100K.
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Old 05-11-2011, 11:52 AM   #3
joewales
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Hi Grassgirl,
thanks very much for replying. I will hold back from continuing onwards for now then given your experience.
I find it strange that the fluorometer reading indicates there is enough cDNA as this is what you base the dilutions on for the next step.
Roche support told me that I could run some out on a gel - I tried this but couldn't see anything. They also said that a common way to lose material is by leaving the beads too long when they are drying - as you suggested.
I asked Roche if we could amplify the cDNA library, using primers for the adaptors, but they said that they can not release the adaptor sequences for some reason.
We are looking into RNA amplifcation kits but they are quite expensive.
I thought maybe I could try redoing the library - I still have mRNA left (just) - and this time taking an aliquot out before the adaptor ligation step. Then proceed otherwise normally to the end with both the aliquot and main sample but missing out the adaptor ligation step for the aliquot. Then clone the aliquot into a TOPO vector, grow in cells, miniprep and do M13 PCR to see what size range I get. I'm not sure that would work though. Also, if the problem is not enough cDNA it doesn't really help besides confirming size range.
May I ask what time you ended up using for the incubation step?
Thanks again,
Joe
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Old 05-12-2011, 02:01 AM   #4
gogreen
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Hi Joe,
I don't work on Roche library preps, but the problem sounds familiar! In my experience, the Ampure beads are very effective in recovering most of the stuff in the reaction, but when one lets them dry too much, the resuspension becomes harder. What do you use to resuspend? TE buffer or EB would work better for resuspension of dry beads.

I would try to re-run a bioanalyzer (High sensitivity, of course!) with higher concentrations. You could also try to PCR amplify (instead of emPCR) an aliquot of the library using the sequencing primers just to see that you have the right size of library or nothing at all. (maybe this one is not possible for Roche library, I am not aware of the process). I've also seen that if you start with more DNA/cDNA in the PCR, the amplification is sometimes very poor.

Cloning an aliquot of the cDNA and post ligated DNA would help you figure out if it was a problem with the cDNA purification or the library prep!
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Old 05-12-2011, 09:41 AM   #5
grassgirl
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Hi Joe,

Well, I'm not sure if overdrying is the problem in my case or not. Possibly. I also have questions about how good Roche's cDNA prep kit is. After looking at another thread, it seems that others have had better cDNA yield with Clontech's Smart cDNA Kit. I am thinking of giving it a try. Here is the link to that thread:

http://seqanswers.com/forums/showthread.php?t=7796

For the fragmentation, I found that 2 minutes worked well, regardless of pH of the fragmentation buffer. I ran a 3-pH, 2-time point experiment. I posted the bioanalyzer results on the following thread so if you want to take a look, here is the link:

http://seqanswers.com/forums/showthread.php?t=10271
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Old 06-28-2011, 07:43 AM   #6
joewales
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Thanks very much for all your suggestions. We have had a long delay waiting for new bioanalyser chips but are now back up and running.
I tried redoing the protocol with some other mRNA (3ug) from another sample just to test out whether the starting material was too low.
I took out aliquots after each stage in the prep and ran them on the high sensitivity chip. It looks like I am losing most of the material in the small fragment removal stage. Does anyone else have this problem? I don't think it can be the bead as they are the same as those used for purifying the cDNA earlier (after second strand synthesis) and that was fine.
Also, does anyone know how the sizing solution works or if there is any way around it? I have asked Roche but am still waiting for a reply.
Thanks again,
Joe
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