8 In Situ Hybridization for RNA: Radioactive RNA Probe
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b) Spot 5 ilion two filters. One of them (Filter A) is used to
measure total count, while the other filter (Filter B) is
washed five times with 5 ml 10% trichloroacetic acid
(TCA), followed by 5 ml of 95% ethanol.
c) Place each filter in the corresponding scintillation vial.
Add 5 ml scintillator.
d) Measure radioactivity (dpm) of each vial.
e) Calculate incorporation ratio (dpm of Filter B/dpm of
Filter A).
9. Add 1111 DNase 1(2 U), incubate at 37 D C for 15 mins.
10. Add 1 III 3M sodium acetate and 1 III Ethachinmate
11. Add 2.5 vol. ethanol
12. Centrifuge 15xg for 5 mins.
13. Remove supernatant and wash with 75% ethanol
14. Dry the samples
15. Remove supernatant carefully by capillary (made by pasture
pipette)
1. To reduce the probe size to alSO rv 300 bp, add 200 III of
attached alkaline hydrolysis buffer.
2. Incubate at 60
0
C for a period of t calculated with the following equation:
t = (Ii - If)/k Ii If
where k = 0.11 Kb/mins, li:initial, If:final Kb.
3. Add 20 III (1M NaOAc pH 6) and glacial acetic acid 1111 (final
0.5% v/v).
4. Add 560 III ethanol and store -20 D C for 1 hr.
5. Spin and wash with 80% ethanol
6. Spin and remove ethanol carefully with capillary.
7. Dissolve the pellet in 25 III of water and add 2.5 III DTT (0.1
M) and 1 III RNase inhibitor).
8. Store at -80
0
C.
Alkaline hydrolysis of
RNA probes