Badly need some help as I did not find any exact protocol for my experiment anywhere, I am working with human platelet to find out the effect of charcoal on the expression of platelet glycoprotein IIIa, IIb and Ia. Now I am facing serious problem in two points. 1. RNA isoltaion and 260/280 ratio 2. qPCR reproducibility in melt curve.Briefly my protocol is: I start directly with PRP, first dilute the 40ml PRP in 280ml normal saline and then take 2 set 10ml diluted PRP one with and another without 10 gram charcoal and shake 30 minute and I filter the PRP using 40 micron filter and collect supernatant and isolate platelet by centrifusing at 1600g for 10 minutes and then collect the pellet as platelet and try to isolate RNA using different techniques first I used qiagen RNeasy mini kit but every time I got only 2-5 ng/μl RNA and 260/280 ratio 1.4-1.5, then I tried trizol reagent this time I got around 100-150 ng/μl RNA but the 260/280 ration is 1.5-1.55 ng/μl and using this RNA I prepare cDNA and did qPCR against GPIIa, IIb And Ia specific primer and GAPDH as reference but I found different melt curve for same primer every time and gene expression is also different in different time sometime expression increased and sometime decreased for the same sample. So I thought RNA extraction and purity problem so I tried trizol+Qiagen RNeasy mini kit combinedly but the RNA was 20-40 ng/μl and 260/280 is 1.5 then I tried another method first isolate RNA by trizol the use RNesy mini elute clean up for purification but found same result. so can anyone kindly give me ant suggestion that how can i get high amount of RNA and 260/280 ratio higher than 1.90 and how can I get the exact qPCR result. Any kind of help will be very helpful for me