Combined microRNA and mRNA detection in mammalian retinas by in situ hybridization chain reaction

Improved in situ hybridization methods for mRNA detection in tissues have been developed based on the hybridization chain reaction (HCR). We show that in situ HCR methods can be used for the detection of microRNAs in tissue sections from mouse retinas. In situ HCR can be used for the detection of two microRNAs simultaneously or for the combined detection of microRNA and mRNA. In addition, miRNA in situ HCR can be combined with immunodetection of proteins. We use these methods to characterize cells expressing specific microRNAs in the mouse retina. We find that miR-181a is expressed in amacrine cells during development and in adult retinas, and it is present in both GABAergic and glycinergic amacrine cells. The detection of microRNAs with in situ HCR should facilitate studies of microRNA function and gene regulation in the retina and other tissues.


Solutions
20X PBS NaCl 160.0 g KCl 4.0 g Na2HPO4 28.8 g KH2PO4 4.8 g Dissolve in 800ml of water. Adjust the pH to 7.4 with HCl. Adjust final volume to 1 Liter. For RNase-free PBS, treat with diethyl pyrocarbonate (DEPC) 0.1% overnight at 37°, then autoclave. DEPC should be added in a fume hood with appropriate protective gear. 20X

microRNA in situ HCR for tissue sections with optional combined antibody staining
Methods for in situ HCR in Fig. 1, Fig. 2, Fig. 4a-l.
All steps are performed at room temperature (~20° C) unless indicated otherwise.

microRNA in situ HCR for tissue sections with combined split initiator mRNA in situ HCR
Methods for in situ HCR in Fig. 4o, p and Supplementary Fig. S3 All steps are performed at room temperature (~20° C) unless indicated otherwise. 10. Wash slides in PBS for 10 min.
11. Pre-hybridize the slides in hybridization solution without probe. ~2.5 hr at 37°C in incubator (precise time for pre-hybridization is not critical). Pre-warm hybridization solution with probe(s) at 37°C for 10 min. before adding to slides.

microRNA in situ HCR for tissue sections combined with two-initiator mRNA probes for in situ HCR
Methods for in situ HCR in Fig. 3, Fig. 4m-n, Supplementary Fig. S2.
All steps are performed at room temperature (~20° C) unless indicated otherwise.

Perform this step in a fume hood with appropriate protective gear.
3. Wash slides twice in PBS for 10 min. each.
4. Treat the slides with 10 microgram/ml Proteinase K in PK buffer for 6 min. (see note 1) 5. Wash slides in PBS for 10 min.

Perform this step in a fume hood with appropriate protective gear.
7. Wash slides in PBS for 10 min.
8. Rinse slides briefly in RNase-free water.
9. Acetylation: Incubate slides in 0.1M TEA + acetic anhydride for 5 min. twice. Immediately before transferring slides to the dish, add 0.5 ml acetic anhydride to 200ml 0.1M TEA (20ml 1M Triethanolamine (TEA) pH 8.0 + 180 ml RNase-free water). Mix with a pipet tip and/or by gentle rocking to disperse acetic anhydride. Transfer the slides as soon as acetic anhydride is dispersed.

Perform this step in a fume hood with appropriate protective gear.
10. Wash slides in PBS for 10 min.
11. Pre-hybridize the slides in 50% FA hybridization solution without probe. ~2.5 hr at 37°C in incubator (precise time for pre-hybridization is not critical).
Hybridize mRNA probes overnight at 37°C for ~18 hours.
16. miRNA probe hybridization using hybridization chamber coverslips: Add 20 pmol of the miRNA probe to 200 µl of hybridization solution (the microRNA probe is at a final concentration of 100nM).
Pre-warm hybridization solution with probe at 37°C for 10 min. before adding to slides.
Hybridize probe overnight at 37°C for ~18 hours.  Fig. S1. (a) Schematic diagram of miRNA in situ HCR. A miRNA probe is hybridized to a target miRNA. The probe includes two initiator sequences, one at each end.
After hybridization and washing to remove unbound probes, the bound probe is detected with a pair of 36 nt HCR amplifiers, both labeled with the same fluor (either Cy3 or Cy5). Each hairpin amplifier that is base paired with a probe initiator sequence in the initial round exposes a free initiator sequence for the other hairpin of the pair. Additional hairpin amplifiers bind to the newly exposed initiators, and the process repeats to generate large polymers of hairpin amplifiers at each probe binding site. If two miRNA probes are used, two distinct sets of hairpin amplifiers, one labeled with Cy3 and the other with Cy5, can be used simultaneously to detect the two miRNA probes independently. (b) Schematic of mRNA in situ HCR using probes with an initiator sequence at each end. Probes are longer than the miRNA probes, requiring different hybridization and washing conditions, but the HCR detection process is the same as in (a). Only one mRNA probe is shown, but pools of up to 5 probes against the same mRNA can be hybridized and detected together to increase signal. For combined mRNA and miRNA in situ HCR, the longer mRNA probe pool is hybridized and washed first, then the miRNA probe is