First-strand cDNA synthesis from paraffin embedded RNA - Using of gene-specific primers (Mar/23/2011 )
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.
It sounds that your purpose is to get mRNA expression from paraffin embedded tissues. It is very challenging to get meaningful data from paraffin embedded tissues even you can manage to amplify small fragments of cDNA. I believe in this case random primers should give you greater chances than gene specific primers for cDNA synthesis.