In situ Hybridization

It is possible, using the technique of in situ hybridization, to identify individual cells expressing a particular mRNA. To do this we need to synthesize an antisense RNA molecule— an RNA that is complementary in sequence to the mRNA of interest. In a test tube, the appropriate strand of the cloned cDNA is copied into the antisense RNA using RNA poly-merase. The RNA is then labeled with a modified nucleotide that can subsequently be detected using an antibody and a color reaction. The cDNA must first be cloned into an

1. Denature RNA

2. Separate RNAs on denaturing agarose gel

1. Denature RNA

2. Separate RNAs on denaturing agarose gel size markers size markers

3. Blot RNA onto membrane filter

3. Blot RNA onto membrane filter

4. incubate membrane with cDNA probe

5. Autoradiography

CYP2B1 mRNA

specific mRNA detected by cDNA probe

5. Autoradiography specific mRNA detected by cDNA probe

a

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