How do I choose the right band for sequencing if multiple components appear in the same or different tissues after the RACE reaction?
There are several scenarios to consider. The first is that no differential splicing occurs at the 5 end of your RNA. If your gene is expressed in more than one tissue, you should choose the largest common band from these tissues. Always choose well-separated bands for sequencing. The second scenario is that alternate transcriptional initiation or splicing exists at the 5 end of the RNA, either in the same tissue or in different tissues. Comparison of different size bands in all the tissues will enable you to determine how many alternatively initiated or spliced transcripts exist. While truncated PCR products may exist in some tissues, differentially initiated or spliced transcripts have defined sizes and they can be found frequently in more than one tissue. To confirm the existence of alternatively initiated or spliced products, duplicate RACE reactions should be conducted using the four plates that are provided.
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