MicroRNAs (miRNA) are short, non-coding RNAs involved in post-transcriptional silencing of gene expression. miRNAs can be associated with exosomes and can function as cancer-specific biomarkers. This, coupled with the fact that they are stable in plasma and serum makes them valuable diagnostic tools, as long as they can be reliably isolated from the serum and plasma.
In this article I’ll give you some top tricks for isolating miRNAs from plasma and serum followed by a Bitesize overview of the protocol.
But first, a quick orientation for newbies. Plasma is the cell-free supernatant produced by centrifuging blood containing an anticoagulant, which prevents it from clotting. If we don’t add any anticoagulant, and proceed to centrifuge blood, we will obtain serum, which is the cell-free supernatant produced from blood that has spontaneously clotted.
So, Here are My Top Tricks for Isolating miRNAs From Plasma and Serum
Trick 1: Select an anticoagulant that is compatible with downstream steps
Whereas EDTA and citrate anticoagulants are both compatible with downstream qRT-PCR steps, heparin can potently interfere with subsequent PCR reactions.
Trick 2: Prevent cell contamination in serum and plasma samples
Cells have a much higher miRNA concentration than plasma or serum, so cellular RNA carryover from cell debris can introduce significant variables into your results and throw off your calculations. Avoid cell carry-over at all costs
Trick 3: Dilute serum/plasma samples with a denaturing solution
Serum and plasma have high concentration of proteins. Therefore, it is common to use denaturing solutions to dilute samples. Usually, ten volumes of Trizol or Qiazol reagent are sufficient to dilute one volume of plasma/serum.
Trick 4: Use spike-in synthetic non-human miRNA control
To adjust for possible variations in RNA extraction and/or RNA amounts, spike-in synthetic non-human miRNAs are frequently used after the sample denaturation step. They also provide an internal reference for normalization of technical variations and recovery efficiency between samples during RT-qPCR.
And Here is an Outline of a General miRNA Isolation Protocol From Plasma/Serum Samples
- Thaw plasma and serum samples completely on ice and mix each sample by inverting, NOT vortexing, the tube to evenly disperse the components.
- Add 10 volumes of Trizol or Qiazol solution to one volume of plasma/serum and mix well by vortexing to effectively denature proteins, then incubate at room temperature for 5 to 10 minutes.
- Add synthetic spike-in miRNAs controls and vortex the sample immediately.
- Add 0.2 volumes of molecular grade chloroform for aqueous and organic phase separation. All RNA, including miRNAs, will be in the aqueous phase, which is the top clear phase. To obtain a stable, clear aqueous phase, homogenize the denatured sample by vortexing at the maximum setting for 30 seconds then centrifuge at 12,000g for 15 min at 4o C. Make sure that the cap on the tube is tightly closed.
- Transfer the aqueous phase containing the total RNA to a new tube, but avoid the interphase, which is a yellowish-whitish band between the aqueous and organic phases.
- Now you can use a number of commercially available kits, to elute the total RNA or enrich for the miRNA fraction according to the manufacturer’s protocol.
If you need to isolate miRNAs from plasma or serum, I hope this gives you a good orientation, and perhaps some good ideas. If you have any comments or questions, please add them in the discussion below!