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initial amount of total RNA or enriched small rna for small rna seq
Hi everyone,
many thanks
We are planing to small RNA sequencing using Illumina Genome Analyzer (Solexa), my question is how much the initial amount of total RNA should be in general ? or enriched small rna?
many thanks
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Which species? And how about the length restriction?
For what we did for human and other mammals at the length range less than 40nt, the most of final results were known miRNAs, about occupied 80~90% or even more. let-7 families were the highest abundant families. -
First, thanks ishmael for your response
We want to sequence two endemic fish species whose genome are not sequenced and annotated in database. Our study mainly covers the microRNA sequencing and their proposed relatively lenght is between 18-30 nt. My question is how much (ng or ug) the initial RNA quantity (small enriched rna or total rna) should be?
thanks...Comment
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Sorry, I forget the topic is about library preparationOriginally posted by Deli Çoban View Post
Guys in our wet lab said it is hard to say the mass of total RNAs, may be 1mg? They just followed the protocols. Sometimes only when the sequencing finished then you know if the exaction is messed up or not. In the beginning, some sequencing batches of our samples were full of adaptor sequences only, useless
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Hi, ishmaelOriginally posted by ishmael View PostSorry, I forget the topic is about library preparation
Guys in our wet lab said it is hard to say the mass of total RNAs, may be 1mg? They just followed the protocols. Sometimes only when the sequencing finished then you know if the exaction is messed up or not. In the beginning, some sequencing batches of our samples were full of adaptor sequences only, useless.
During the library preparation, approximately 1 ug (< 50 pb) enriched small RNA sould be suffecient to get proper sequences for illumnia hiseq2000, according to friends who runs this machine routinely. But, now I embark on collecting part of article about my subject. And, I am planing to list this information here.
kindly...Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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