5 um filter tube

[katinka]

HI all, I would like to ask about your experience with small RNA Seq (TruSeq Small RNA Library Prep)/MiSeq, especially the part when the cDNA library is eluated and spun down through the 5 um filter tube. Do you have any expreiences with alternative solutions when it comes to 5 um filter? Any other provider than IST Engineering, thawing the gel instead and so...

Thank you in advance

[ENT Fellow]

Shortly will have some answers

I am in the process of ordering these parts (not easy) and hopefully will be able to give you some answer in about 2 weeks

[katinka]

Hello, thank you a lot...finally I did some experiments with eluting some cDNA of very similar size as the library. Well, to make the eppendorfs with small holes to put the gel through, perfect, no problem, removing the gel just by pipetting was a bit awfull, but worked as well. Finally I ordered these parts as well, but you will have the experience much sooner, hope everything will go well for you.

[ENT Fellow]

thanks for that - Can I ask about the starting amount of RNA you have used? I'm using FFPE tissue samples and have only a small amount so I was going to try starting with whatever I get left after depleting the ribosomal RNA from 500ng of total RNA. I suspect this is going to leave me with some significant adaptor contamination so I was thinking about diluting the adaptors down to a 1 in 10 solution. What sort of starting amounts have you used? (and did you deplete the ribosomal RNA first?)

[katinka]

hi, sorry for not responding...actually - the first question - I wasn't and am not planning to get rid of the ribosomal RNA now (we'll see, but when using the SOLiD sequencer, it didn't cause any bigger trouble, so hope it will work ok again). When it comes to the amount, I am planning to start with 1 ug as recommended. Actually NEBNext kit can be used with smaller amount...as for work with FFPE samples I'd be more nervous about the RNA quality, how are your RINs anyway and so?...but guess that smaller amount will work for you as well...

[ENT Fellow]

Ahhhh - you're on the SOLID sequencer - we use the Illumina HiSeq. I've used the NEB kit with FFPE RNA before - sequenced it and got quite a bit of ribosomal RNA contamination on alignments, so I've changed to the Illumina kit after speaking to a few other people and am definitely depleting the ribosomal RNA. I have no experience of the SOLiD so may work better. FFPE RNA doesn't really give you a RIN number but we've managed to do CNV, exome, and transcriptome Sequencing from it with near identical results when compared to fresh frozen samples so I'm hopeful we can conquer miRNASeq

[katinka]

Well, I used the SOLiD once and am happy to start working with Illuminas MiSeq and actually I have some data from HiSeq as well but these were done for me by a cooperating group, so don't actually know the troubles they faced...