Hi everyone, I was wondering if I could please run something by you all and gain some feedback? 

I am required to synthesise cDNA from RNA for qPCR experiments, so far we've been able to get a good quantity of RNA from our tissues and generate 1000ng in a 20ul cDNA synthesis reaction. The kit I use is the Transcriptor First Strand cDNA synthesis kit which is available from Roche; after adjusting component volumes in a 20ul reaction, we are left with 10ul in which to add enough RNA to make up to 1000ng, and water to fill the remaining volume.

However I've been working on another set of samples and the RNA that I obtain from these tissues is much lower than before; there is not enough to make up to 1000ng in a 20ul cDNA reaction e.g.) 51.3ng/ul * 10 = 513ng - and the remaining 10ul of the reaction must be made up with the other components such as dNTP's e.t.c

I was wondering if it could therefore be possible to increase the reaction volume from 20ul so I can add more RNA to make up to 1000ng? e.g.) If I prepared a reaction volume of 40ul I would double the volumes of the cDNA components, and according to the Roche protocol this would leave me with 20ul in which to add RNA and water. If I used my example from before: 51.3ng/ul requires 19.49ul to make 1000ng, I'd then need to add 0.51ul of water and the rest of the components to make up to 40ul.

Is this how the procedure would work?    

Or, due to the increase from 20ul to 40ul, would this further dilute the newly synthesised cDNA? 

I look forward to your answers, many thanks in advance!