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Old 08-12-2010, 03:45 AM   #1
bpa400
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Question Problem with small RNA library preparation

Hi,

I'm hoping someone can provide me with some answers regarding the small RNA library preparation.

I have now used both the Illumina small RNA v1.5 and the Bioo Scientific AIR small RNA sequencing kit. However, I only ever end up with adapter dimer and no small RNA bands around 100 bp. I have cut the gel at 100 bp and run it on a DNA chip on the Agilent Bioanalyzer to confirm that there is nothing there. I also find that with the Bioo Scientific AIR kit, there is significantly less adapter dimer and the band tends to be tighter than with the Illumina kit.

I am preparing samples from total RNA extracted from leukocytes using the trizol method and have tried concentrations ranging from 1 to 10 g. Despite this, every time I prepare a library, I end up with an adapter dimer band (with the Illumina kit, this runs to almost 100 bp, the Bioo Scientific kit it is much tighter around 75-85 bp) and no small RNA library. At this point the obvious question is whether or not I have small RNA's in my samples. However, I am convinced that I do and have validated this on gels and using the Agilent RNA chip. I know the 3' adapter is supposed to be pre-adenylated to bind specifically with the small RNA fragment of my sample but how specific is this as it doesn't seem to happen in my sample?

I'd appreciate any advice if there is anyone else who has had this issue.

Many Thanks!
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Old 08-12-2010, 06:56 PM   #2
link1
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sounds like your sample might be lacking small RNA? I would recommend running your sample on a small agilent chip:
http://www.chem.agilent.com/Library/...G_smallRNA.pdf

make sure its the small chip you run. you might be losing the smalls in your trizol extraction. on a gel you won't be able to see the micrRNAs.
I've made ~20 small libraries with both vendor's kits with success so far. In the beginning I was losing the smalls in my trizol extraction. Using the small chip helped me identify which trizol preps were good and which were bad.
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Old 09-23-2010, 03:28 AM   #3
bpa400
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Quote:
Originally Posted by link1 View Post
sounds like your sample might be lacking small RNA? I would recommend running your sample on a small agilent chip:
http://www.chem.agilent.com/Library/...G_smallRNA.pdf

make sure its the small chip you run. you might be losing the smalls in your trizol extraction. on a gel you won't be able to see the micrRNAs.
I've made ~20 small libraries with both vendor's kits with success so far. In the beginning I was losing the smalls in my trizol extraction. Using the small chip helped me identify which trizol preps were good and which were bad.
I have run some of my samples on the Agilent small RNA chip now. The electropherogram shows a slight bump between ~10 - 30 nt and I'm getting micro RNA concentrations between 150-750 pg/ul. Is this a sufficient amount of micro RNA for the library preparation? Would reducing the adapter concentrations help to reduce adapter dimer while increasing the amplification efficiency of my small during PCR?
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Old 11-01-2010, 02:56 PM   #4
pbluescript
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If you are not already, make sure you add your ligase to the reaction last. I have seen that cause problems with adapter dimer before, especially with low input samples. Lowering the adapter concentrations would help as well.
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mirna library, mirna seq problem, problem, rnaseq, small rna

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