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Template Generation
| Gene expression is detected with digoxygenin-tagged RNA probes that are generated from DNA templates. Such templates are usually synthesized by RT-PCR. Using sequence databases, templates in the range of 1000 bp are designed. Two primers consisting of 25 nt of gene-specific sequence linked to SP6, T3 or T7 polymerase promoter sites are used to amplify the desired gene from a cDNA pool (representing a variety of embryonic and postembryonic tissues). Initial PCR products are gel-purified, sequence-verified, amplified and template DNA is used for in vitro RNA synthesis in the presence of digoxygenin-tagged UTP. |
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