miRNA Extraction from Plasma

[DrDTonge]

Dear all,

I'm currently working on the isolation of miRNA from canine and human plasma samples. Using the Qiagen column based protocol, I have been able to isolate around 300pg/ul of miRNA (based upon a 4-40nt region using Agilent small RNA chips) although I understand this is insufficient to proceed to library preparation. My overall aim is to use SOLiD to determine differentially expressed miRNA (both known and novel) from clinical plasma samples of <= 1ml.

I wonder whether anyone else has had any more success in obtaining a higher miRNA yield, or whether anyone could recommend any amplification methods / isolation protocols? To date, I've not had much luck in finding any publications to guide us.

Best wishes,

D

[aushev]

We had the final concentration similar to yours. In some papers, authors claim much higher yield but I have an impression that it is just due to wrong S/F quantification.
I never tried to make library for sequencing, but - maybe your amount will be still sufficient? In the protocol, chapter "Prepare Small RNA Libraries", it is said "If you started with enriched/purified small RNA and input 1–10 ng, run 18 cycles." 10 ng corresponds to 33 mkl of your product - which seems doable, doesn't it?

[ECO]

Moving to Sample Prep

[DrDTonge]

We had the final concentration similar to yours. In some papers, authors claim much higher yield but I have an impression that it is just due to wrong S/F quantification.
I never tried to make library for sequencing, but - maybe your amount will be still sufficient? In the protocol, chapter "Prepare Small RNA Libraries", it is said "If you started with enriched/purified small RNA and input 1–10 ng, run 18 cycles." 10 ng corresponds to 33 mkl of your product - which seems doable, doesn't it?

Dear Aushev,

Many thanks for your response. I agree, the higher concentrations are likely a result of inaccurate quantification although it's always nice to hear someone else is getting similar yields to ourselves!

With regards small RNA preparations, yes, it is doable although we were trying to gain enough sample to run both deep-seq and miRNA array from the same extract. I am going to run the library preparation in the next week or so, so will report on any progress.

Many thanks once again,

D

[aushev]

With regards small RNA preparations, yes, it is doable although we were trying to gain enough sample to run both deep-seq and miRNA array from the same extract. I am going to run the library preparation in the next week or so, so will report on any progress.

Many thanks once again,

D

I will be very thankful to hear any news about your experience on deep sequencing of plasma miRNA as I will probably try to do the same (also on SOLiD) in future.

Good luck and all the best for your experiments.

[vivekk]

Hello,

I have question about your experience with miRNAs in plasma so far. When you sequenced for miRs, did you get an estimate of what kind of depth you need to enrich / detect most miRNAs in your sample?

I know that with tissue samples one can detect most miRNA content with anywhere from 5-10 million reads/sample. Anyone know / have an idea for plasma? Would 500K - 1M reads / sample be a decent depth?

Thanks,
Vivek

[brendonp77]

I am also curious whether anything up to 2 million reads is enough for plasma derived small rna...i feel the standard 5-10 million reads is much. With qiagen mirneasy the percentage of 4-40nt abundance assessed by agilents small rna quant kit is about 30%.

[JQL]

I am currently looking into miRNeasy serum/plasma kit for dog serum samples. It claims that RNA from serum and plasma typically consists of molecules <100 nucleotides. Purified RNA can then be used for biomarker discovery with the miScript PCR System.

Does anyone here use the kit? Any additional enrichment/purification necessary? I am going to do Illumina seq. thank you for your opinion.

thanks

[blanco]

Hi all who have some experience/knowledge in working with RNA from plasma.

I just did some extractions and was deterred when I ran the RNA Nano on the bioanalyzer and found no trace of RNA.
I then ran the samples on the 'small RNA' bioanalyzer chip and this time we were able to pick up some RNA.

Attached below is one of our 'good' results.


So, I have a couple of questions:
Is this trace what one would expect from plasma RNA?
And, is this sufficient amount of RNA to construct a 'small RNA' library?

Any kind of help or comments would be appreciated.
Many thanks,
blanco