I have two basic questions about the use of gene-specific primers in the first strand cDNA synthesis:
1. What limits the fragment length?
2. In this case more amount of RNA template is necessary, right?
The RNA I have is from paraffin-embedded sections.
I found a detailed protocol about how to use gene-specific primers for first-strand cDNA synthesis (I put it here, if someone is interested) and the authors say they just apply several reverse primers to the RNA-template and other reagents.
The fragments they generate are longer than mine. When I use the random hexameres, afterwards I can amplify the desired fragments. In this case, shall I expect that generation of my fragments of interest is possible?
I will appreciate any additional advice.