Weak RT-PCR products? - (Sep/03/2004 )
I've been trying to obtain good RT-PCR products from weakly expressed transcripts for a while now, my cycle numbers are as low as they go and I've increased the concentration of cDNA in my PCR step etc... but the products are still very hard to see. So, my next step might be southern blotting to enhance the visibility of my bands as I think I'm seeing the right expression patterns (then again, it could be bad eye-sight and some wishful thinking!). I've come across one protocol describing southern blotting using cDNA and PCR product from genomic as probe, has anyone tried this and how does it turn out? Otherwise I shall try simply running out my RT-PCR products themselves and probing with PCR products?, any advice greatly appreciated!
I know nothing about southern blotting of cDNA, but I think PCR is the most sensitive method and its theoretic limit is one copy of your template. You may want to opitmize your PCR conditions before you abandom PCR.