Small RNA Library Question - SOLiD

[DrDTonge]

Dear all,

So I've prepared my first smallRNA library from 4 fibrotic skins samples and 1 plasma sample. Following reverse transcription and amplificiation, I have run an Agilent DNA chip to determine the % of ligated small RNA to total RNA in each reaction. These figures have fallen below the threshold required to progress to bead prep (50%) with the best being around 30%.

From the traces, it seems that I have a predmoninance of small products (too small to represent miRNAs with 3' and 5' adapters) with only between 10 and 30% of the sample being of the desired size.

The manual recommends to perform a second round of size selection at this stage (on the resulting amplified PCR products) using PAGE to excise just the size indicative of miRNAs plus adapters.

My question is, would it not be more useful to use a small amount of the resulting PCR product in a second round of amplification?

Your help would be most appreciated,

D

[DrDTonge]

Dear all,

Just for future reference, I've tried the above technique (re-amplifying a small amount of the amplified cDNA using SOLiD 5' and 3' BC in an attempt to increase the % of miRNAs with adapters) with no success.

I'm therefore going to procede with a further round of size selection.

Best wishes,

D

[DrDTonge]

Sorry the above was a rather vague (pre-coffee infusion) answer....!

By no success I mean on running the re-amplified product against the original product (Agilent DNA 1000) there was no increase in the % miRNA with adapters. In fact, very little amplification took place during the second round.

Just for reference so you don't waste time repeating what he been tried!

D

[pmiguel]

Hi D,
Also any time you "use a small amount of the resulting PCR product in a second round of amplification", you have the potential to "bottleneck" your library. That is, only the molecules present in your "small amount" can be amplified. So you would have capped the diversity of your library at the number of different molecules in the "small amount".

--
Phillip

[whw]

Do you think those "small products" are adaptor dimer? I had a similar problem making a small RNA library with Bioo Scientific adaptors (for illumina). They suggested adding the first strand RT primer immediately after 3' adaptor ligation. The primer binds any excess adaptor and prevents it from being ligated to the 5' adaptor. Worked great and the results were clearly visible on the bioanalyzer. Hope that helps.

[SeqAA]

They are most definitely adapter dimer. It's a common problem for sRNA preps.

[DrDTonge]

Yeah, I'll second that. Most certainly adapter dimers from Agilent trace. Very interesting point raised by whw...I'll look into this for any future preps.

D