Original Protocol for Fluorescent RNA In-situ using Tyramide Signal ...

Gavin WilkieIlan Davis Lab, Biochemistry 11. Mount embryos in 80% Glycerol or Vectashield and seal the slide with clear nail varnish. Keep slides at 4°C in dark when not in use. The Fluorescence is covalently deposited at the site of HRP reaction and so should not diffuse away over time. REAGENTS REQUIRED TSA kit supplied by NEN Life Science Products (UK 0800 896046) ; also see http://www.nenlifesci.com for more info.) We use the Cy3 kit (NEL734A) but kits for Cy5, fluorescein, rhodamine and coumarine labelling are also available. TNB is 0.1M Tris (pH7.5), 0.15M NaCl and 0.5% Blocking reagent from NEN kit. PTW is PBS + 0.1% Tween20. Hybe solution: 50% deionized formamide 5X SSC 100 µg/ml E. coli tRNA (Sigma) 50 µg/ml heparin 0.1% Tween 20 pH to 6.5 with conc. HCl Making Probe: Transcribe RNA probe using Boerhinger Mannheim RNA Dig labeling kit. The template can be from miniprep DNA (E.coli RNA is o.k.) and must be linearised with an appropriate restriction enzyme (for sense or anti-­‐sense probes) and enzyme heat killed. Use about 1 µg of digest (no more than 2.5 µl) in 20μl probe reaction) Transcription reaction mix: Linearised template (0.5mg/ml) 2μl NTP labelling mix (tube 7) 2μl 10x buffer (tube 8) 2μl RNase inhibitor (tube 10) 0.5μl ddH2O (DEPC) 11.5μl RNA polymerase (T7/T3/SP6) (20U/ml) 2μl Incubate at 37° for 2h. Stop reaction by adding 2µl of 0.2M EDTA. Check yield on gel: load 0.5-­‐1μl before and after incubation. Should see strong novel RNA band as reaction should yield about 10μg DIG labelled RNA.