RNA Detection

Note 5). Each ShortCut reaction in 50μL volume includes

20 μg RNA, 4μL ShortCut RNase III, 5μL 200 mM MnCl 2 ,

and 5μL ShortCut buffer. The reaction is incubated at 37C

for 20 min.

7. After ShortCut digestion, purify RNA using the standard TRI-
   zol method and resuspend RNA in 15μL water. Determine
   concentration of the samples by spectrophotometer and ana-
   lyze size distribution using Bioanalyzer (Fig.2d). Typically, the
   AMT cross-linked samples have a stronger tail above 100 nt
   than the control samples.

3.3 Purification
of Cross-Linked RNA
by 2D Gel

1. Prepare the 12% 1.5 mm thick native first dimension gel using
   the BioRad Mini-Protean 3 gel cassette (Fig.3a). The 1.5 mm
   thick gel is used here to facilitate the removal of air bubbles
   from the second dimension. Use 15-well combs so that each
   lane is narrower and the second dimension has a higher resolu-
   tion. After the gel solidifies, pull the comb very slowly to avoid
   the deforming well dividers.


2. To each 15μL sample add 5μL6Orange G loading dye.
   Load 3μL dsRNA ladder as molecular weight marker. Run the
   first dimension gel at 100 V for 70 min in 0.5TBE. Orange G
   should be 4/5 way to bottom. Usually we have a starting
   current of 15 mA and a starting power of 1.5 W.


3. After electrophoresis finishes, stain the gel with 2μL SYBR
   Gold in 20 mL 0.5TBE, incubate for 5 min. Image the gel
   using 300 nm transillumination (not the 254 nm epi-
   illumination, which reverses the psoralen cross-linking). Excise

First dimension

Second dimension

Pour second dimension

gel from here

First dimension

30-150 bp

1D: native

2D: denatured

–AMT +AMT

A B C

Fig. 3Diagram and example result for 2D gel purification of cross-linked and digested RNA. (a) First dimension
native gel. Gel slices containing RNA around 30–150 bp are usually cut out from the first dimension for the
second dimension. (b) Second dimension denatured gel. Gel slices are aligned between the glass plates
before pouring the urea denatured gel solution at the bottom of the gel plates (as indicated by thearrowin the
middle). (c) An example second dimension gel. Theyellow-boxed areaindicate the cross-linked RNA to be
extracted for library preparation

PARIS: Psoralen Analysis of RNA Interactions and Structures 69