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
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
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