Hybridization

1. The labeled probe is dissolved in water to give approx 20,000 cpm/pL. Ideally, this should be between 0.05 and 0.005 pmol of probe.

2. Between 5 and 500 pg of RNA is taken from storage, dried down (see Notes 7 and 13), and resuspended in 24 pL deionized formamide. Include controls (see Note 6).

3. In a sterile Eppendorf, mix together the 24 pL of RNA, 3 pL of probe in water, and 3 pL of 10X hybridization buffer (see Note 3).

4. The tube is capped firmly, and the tube placed in a boiling water bath for 2 min.

5. After denaturation, the samples are incubated at the chosen incubation temperature (see Notes 3 and 14) overnight. It is important not to let the temperature of the tubes fall below the overnight incubation temperature (see Note 15).

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