14 In Situ Hybridization for RNA: Nonradioactive Probe: RNA Probe
197
template DNA (l1lg/1l1)
lOx NTP mixture
lOx transcription buffer
nuclease-free water
RNase inhibitor
Vortex
Add RNA polymerase
Tip the tube, and incubate at 37°C for 2hrs.
1111
2111
2111
12111
1111 (20U)
Confirm the reaction by gel electrophoresis.
Purification of cRNAs
1. Add 1111 ofO.5M EDTA, 2111 of 4M LiCl, 1111 of lOmg/ml E. coli
tRNA, 75111 of prechilled 100% ethanol. Mix well.
2. Stay at -70°C for 2hrs or at -20°C overnight. Microfuge at
14,000rpm for 15 mins. Discard the supernatant. Rinse the
precipitate with 70-80% ethanol. Dry up the precipitate.
Sizing of cRNAs
1. Reduce the length of cRNAs to 100-150 bases to so that they
can permeate tissue sections.
2. Add the following in the tube:
- 95111 of alkaline solution (63mM Na2C03+42mM NaHC03)
- 5111 of O.IM DTT.
3. Stay the cRNA solutions at 60°C. The reaction time (T) is calculated as follows: T (min) = (La - Lf)/KxLoxLf' La, length (kb)
of cRNA before treatment. Lf, length (kb) of cRNA after treatment. K=O.ll