I used two negative controls. First negative control was igG and second negative control was IP without any antibody. In both negative controls, I got an amplifications after RT. Any suggestions to reduce nonspecific binding?
You can try blocking your beads with yeast RNA. We block our beads for 1 hour at 4C in PBS+0.05% CHAPS (or you can use a non-ionic detergent like Tween-20), and include 0.1 mg yeast RNA per 100 ul of beads.
If your beads do not come pre-blocked with BSA, you may also want to include 1% BSA in the blocking solution. This will reduce nonspecific protein binding.
Abcam has some nice tips for IP troubleshooting (see link below).
http://www.sigmaaldrich.com/catalog/product/sigma/r6625?lang=en®ion=US
http://www.abcam.com/protocols/immunoprecipitation-troubleshooting-tips
Thanks for your suggestion. I tried with 0.5% BSA, still I got some non-specific amplification. As you suggested, I will try using 1% BSA and Yeast RNA.
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