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-   -   initial amount of total RNA or enriched small rna for small rna seq (http://seqanswers.com/forums/showthread.php?t=11551)

Deli Çoban 05-23-2011 08:52 AM

initial amount of total RNA or enriched small rna for small rna seq
 
Hi everyone,

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

ishmael 05-29-2011 11:43 PM

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.

Deli Çoban 05-30-2011 01:35 PM

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...

ishmael 05-31-2011 10:39 PM

Quote:

Originally Posted by Deli Çoban (Post 42791)
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...

Sorry, I forget the topic is about library preparation:D
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 :(.

Deli Çoban 06-04-2011 04:47 PM

Quote:

Originally Posted by ishmael (Post 42919)
Sorry, I forget the topic is about library preparation:D
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 :(.

Hi, ishmael

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...


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