3.3 Size Selection for <200nt RNA using MirVana Kit (see Note 4)

Raise volume of total RNA solution from step 13 of section 3.2. to 150 μL by adding nuclease free water.

Prepare two columns from MirVana miRNA Isolation Kit following the manufacturer’s instructions.

Preheat Elution solution to 95°C.

In a fume hood, add 5x volume of Lysis/Binding Buffer containing the denaturant guanidinium thiocyanate, a potential hazard, (750 μL to 150 μL of RNA suspension) and briefly vortex.

Add 1/10^th volume of miRNA Homogenate Additive (90 μL to 900 μL from the above step) and briefly vortex.

Place on ice for 10 minutes.

Add 1/3 volume of 100% ethanol (330 μL to 990 μL from above step) and vortex briefly.

Filter the entire sample from the step above through one column by adding 700 μL at a time and spinning 5,000 RCF for 1 min. Repeat until the entire volume has been applied to the column. The flow-through contains the <200nt fraction (see Note 5).

Combine flow-through (~1220 μL total) and split into two nuclease-free microfuge tubes (~610 μL each).

Raise the ethanol concentration of the flow through to promote column binding of the smaller RNA by adding 2/3 volume of 100% ethanol (~406.6 μL) to each tube and vortex briefly.

Load the contents of both tubes on a single fresh column by repeatedly adding 700 μL at a time and spinning for 1 minute as above. The flow through can be discarded each time because the <200 nt RNA is bound to the column by this step.

Wash the column with 700 μL of Wash 1 followed by two washes of 500 μL Wash 2/3 each and spinning as above according to kit instructions.

To dry the column, spin empty at 10,000 RCF for 1 min.

Transfer column to clean, low-binding, nuclease-free tube and add 50 μL warmed Elution Solution provided in the kit

Incubate for 2 minutes at room temperature before spinning as above to elute the RNA.

Repeat steps 14–15. Final volume of <200 nt RNA is 100 μL.

Expected yield at this step is 20–30% of the amount of input RNA (expect 12–15 μg of <200 nt RNA).