ultralow microRNA library prep

[deepbiomed]

Hi,

Can anyone suggest a library prep kit for illumina hiseq/miseq, which can work with ultralow amount of microRNAs (50 -200 pg) ? or is there any method to amplify whole microRNAome ?

Best
Deep

[kerplunk412]

Hi Deep,

If your sample is all/mostly miRNA the NEXTflex Small RNA Sequencing Kit v2 from Bioo Scientific should work. We have had good results lately preparing libraries from as little as 10 ng of human brain total RNA with this kit, and there is presumably less than 200 pg of miRNA in 10 ng of total RNA. Please feel free to PM me for more information. As a disclaimer, I work for Bioo Scientific.

One thing you should keep in mind is that a couple of studies have shown that additional PCR cycles introduce negligible bias into small RNA libraries, especially in comparison to the ligation steps, so usually you should be safe using more PCR cycles than the manufacturer recommends. The downside is that eventually you will end up with a library that is mostly adapter-dimer and weird adapter artifact products if you use too many cycles with too little starting material. Experimenting with adapter dilutions may help in that case.

Also, although I don't know much about whole miRNAome amplification it seems conceptually very similar to library prep, so I guess you will run into some of the same input limitations.