10 In Situ Hybridization for RNA: Nonradioactive Probe: ss cDNA Probe
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method (Chomczynski et al. 1987) and then reverse transcripted
as follows.
1. Mix the following in PCR tube:
- Total RNA 1 flg
- 100 flg/ml random hexermer 0.5 fll
- DDW up to 11 fll
2. Add two drops of minseral oil and incubate 5 mins at 60°c'
3. Chill the sample tube in ice water.
4. Mix the following on ice, add to the sample under minseral oil
and pipet it well:
5X reverse transcription buffer
5 mM dNTP mix
0.1 M DTT
M -ML V reverse transcriptase
4 III
2 III
2 III
1 III (200 U)
5. Set the sample tubes on the thermal controller and perform
the following steps:
60 mins
5 mins
3 mins
6. Store at -20°C until usage.
Asymmetric PCR
Two-step asymmetric PCR is performed as follows, in order to
produce sense or antisense ssDNAs.
To provide ds cDNA as a template of the second PCR, the first First PCR
PCR was carried out using the standard PCR conditions with a
pair of primers.