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[cancerous]

Hi all,

I have some genomic DNA samples which were extracted from tissue sections by laser capture microdissection (LCM), so basically their amount is very little (around 10-50 pg/uL). I tested qPCR efficiency with some positive DNA samples of similar and higher concentrations using Platinum 2x Supermix-UDG (Invitrogen) (conc. = 10pg/uL, 1ng/uL and 10ng/uL, i.e. 1x, 100x and 1000x).

I used 2.5uL DNA (amount = 25pg, 2.5ng and 25ng) for total rxn volume 25uL. After the rxn (50 cycles), the result was so frustrating.

Only 25ng sample showed decent Ct values (about 25) but 2.5ng sample showed very high Ct (~38). 25pg sample just showed noise like negative controls. My real samples may show almost like 25pg positive sample but, based on the result, the qPCR system is not sensitive enough.

The protocol sheet of this Platinum reagent says "use 100pg - 1ug gDNA", so I think maybe this is why. Does anyone know qPCR mastermix with better efficiency? or better way to increase the efficiency?

I am considering to change the annealing and extension temperature from 60/60C to ~55/72C. I have not seen any qPCR cycles using temperature other than 60C, so I am not sure if I should do it or not.

Please help me!