cDNA synthesis ASAP - gene expression (Jun/23/2006 )

I run into some problem. I have some rna that is extremely diluted 4ng/ul. I want to make an RT with at least 200ng of RNA which means I would have to use 50ul of RNA alone plus oligo dt, dntp enzyme dtt. I usually have RNA at 100ng/ul and have no problem using 2ul of RNA and doing a 20ul RT reaction:


Usually my protocol looks like this

RNA: 2ul
OligoDT: 1ul
DNTP: 1ul
Buffer: 4ul
DTT:2ul
Enzyme:1ul
H20: 9ul

total 20ul


what do I do now that I'll need to use 50ul of RNA alone? Do I still use the same ammount of reagents with exception of the buffer to make it my final concentration 1x? Or do I have to incrase everything linearly?

ANy info and imput is greatly appreciated.. hope to get some cDNA going today Friday )

cheers

You can use less than 200ng of RNA and still have a quality cDNA prep. I've gone all the way down to ~80ng RNA/cDNA reaction out of necessity without any major problems, but the gene I was interested in was highly expressed. Make sure to qualify your prep by using the proper controls.

what's your total volume? can you precipitate the sample in order to concentrate, then re-measure the concentration and add a smaller amount

You may also try the RNeasy MinElute Clean-up Kit, which also concentrates RNA; however, you will probably lose about 20% of your starting total RNA.