RNA Detection

2 Materials and Solutions

All solutions are prepared using reverse osmosis water and filter-

sterilized prior to use. Aliquots are kept frozen at 20 C and

working solutions are stored at 4C.

2.1 Reagents and
Buffers

1. 100% ethanol.


2. 3% hydrogen peroxide.


3. 100% CitriSolv (Fisher Scientific): CitriSolv is a d-limonene-
   based solvent and clearing agent used for histology. It dries
   slowly without residue and will not harden or shrink tissue. It is
   a safer alternative to xylene.


4. 1PBS: To make 1 L of PBS, dissolve 8.0 g NaCl, 0.2 g of
   KCl, 1.44 g of Na 2 HPO 4 2H 2 O, and 0.2 g of KH 2 PO 4 in
   800 mL water. Adjust pH to 7.4 with 10 N NaOH. Add
   water to a final volume of 1 L.


5. RNAScope kit with AMP1-6, AP-Fast Red, and HRP-Green
   buffers for chromogenic detection of the targets (ACDbio).


6. Custom probes against the target RNA species (ACDbio,see
   Note 1).


7. Tissue fixation buffer: 4 w/v % PFA, 4 w/v % sucrose,
   0.12 mM CaCl 2 , 0.08 M Na 2 HPO 4 and 0.02 M NaH 2 PO 4
   pH 7.4. Prepare 100 mL stock solutions of 1 M CaCl 2 , 0.2 M
   Na 2 HPO 4 , and 0.2 M NaH 2 PO 4 , with the latter two solutions
   at pH 7.4. Combine 8 g sucrose, 24μL 1 M CaCl 2 ,77mL
   0.2 M Na 2 HPO 4 , and 23 mL 0.2 M NaH 2 PO 4 into ~180 mL
   of water. Slowly heat and stir the solution in a hood. Gradually
   add 8 g PFA until completely dissolved. Adjust the volume to
   200 mL with water and filter-sterilize with a 0.45μm bottle top
   filter unit. Store aliquots of 15 mL at 20 C indefinitely.
   Working solution is stored at 4C and is good for 1 week.


8. Sucrose equilibration buffer: 30 w/v % sucrose solution in
   water. Weigh 150 g sucrose and dissolve to a final volume of
   500 mL of water. Gentle heating and stirring aids in dissolving
   sucrose. Filter-sterilize with a 0.45μm bottle top filter unit.
   Store aliquots in 50 mL and freeze at 20 C.


9. Antigen retrieval buffer: To unmask epitopes made unavailable
   by fixation, make a solution composed of 10 mM citric acid,
   0.05% Tween 20. Add 1.92 g anhydrous citric acid in a 1 L
   beaker and dissolve it in water. Adjust the pH to 6.0 with 10 N
   NaOH. Add 0.5 mL 100% Tween 20 and adjust volume to 1 L
   with water. Store at room temperature for 3 months. Proper
   pH is critical for efficient antigen retrieval.


10. Tissue Freezing Medium (TFM) (Polysciences): A support
    matrix for embedding tissues for cryosectioning that freezes

ISH Detection of MicroRNA and Target Gene mRNAs 199