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 µg. 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!
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 µg. 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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