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Old 09-20-2012, 04:57 AM   #8
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
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Quote:
Originally Posted by rubyryan View Post
Thanks for that! Further to this, once I have obtained the ideal total RNA concentration, (200 ng/ul) I am going to try and sequence all of the small RNA (ie microRNA and piwiRNA) using the Illumina TruSeq small RNA library preparation and then sequence on the HiSeq Illumina. Is this feasible or is it better to focus on one family of small RNA?
Thank you
The Zymo columns mentioned up-thread seem to work well.

If you are using the Illumina TruSeq small RNA prep kit, then there is no need to isolate small RNA. It is designed for 1 ug of total RNA substrate. Even the DNAse treatment may not be needed -- it is recommended so that the amount of total RNA can be accurately estimated. But if you have a fluorimeter, like a Qbit, you could just use an RNA-specific fluor to determine your RNA concentration.

We have sequenced a larger "window" of small RNA sizes. Ended up running them in a 100 nt run, because no other 50 nt samples were in the queue. Looks like we got sequence of both miRNA and some other (larger) RNAs. Analysis isn't back yet, but I guess it worked.

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Phillip
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