FISH

Whole mount in-situ hybridization starts off with two independent procedures that eventually converge: probe generation and preparation of embryos. The entire procedure becomes one during the hybridization step, with I will describe in greater detail.

Probe generation will allow me to synthesis the antisense RNA probes that will bind the alpha-4 gene and help visualize gene expression. The step begins with obtaining the cDNA of the alpha-4 gene. Since the cDNA has been obtained by reverse transcription of mature alpha-4 mRNA, the issue of having introns within the sequence is circumvented. The polymerase chain reaction (PCR) of the cDNA sequence will allow me to amplify the sequence and introduce the RNA polymerase promoter. After PCR, I will purify the resultant sequences before performing an in-vitro transcription to synthesize the antisense RNA probe. Once I’ve made the RNA probes, all further steps will be performed after the application of RNAse-away on all surfaces to avoid probe degradation; RNAse degrades RNA molecules. I will also use RNAse-free water and filter tips as further precautionary measures. Observing the necessary precautions, I will label the RNA probes with digoxigenin uridine-5’-triphosphate, which will enable visualization of alpha-4 gene expression alter on.