I have RNA from microdissected samples that I need to amplify so I will have enough material to test all of my genes of interest in qPCR. Does anyone have experience with the kits out there and can you recommend one? They are very expensive and I do not want to choose the wrong kit.

Thank you!

Tipone,

Are you looking to make cDNA from your RNA? or truly amplify the RNA such as for Microarray analysis?.

If you're asking about cDNA amplification. I use the Invitrogen SuperScript III kit pretty much weekly with excellent reproducible results in qPCR with as low as 0.1ug of RNA.

If you're talking RNA amplification you may want to read this page

http://folk.uio.no/vigdisny/RNAampindex2.html

which has tons of info on it.

let us know what you decide

Rb

I truly would like to amplify RNA. We are studying ion channels in neurons from gut tissue and from the 200 cells I can microdissect I do not end up with enough cDNA to run qPCR for my 12 genes of interest and the HKG.

Do you recommend that I purchase the reagents separately or buy a kit? I figured that a kit would be validated (hopefully) and save me some time.

-T

I realise that the amount of RNA present in a cell depends greatly on the cell type, but Roche have information that suggests that it should be possible to recover 20 microgramms RNA from 1 million cells. If this is true then your 200 cells could contain 4ng RNA. You might need to use carrier (glycogen of tRNA) but Stratagene have a cDNA kit that is supposed to be able to make cDNA from 1ng RNA:

I've just had a look at their product info and they show data for 50 cell samples. The products are:

RNA prep: SideStep mRNA Enrichment Kit
cDNA prep: StrataScript RT

I usually get about 2 ng total or so of RNA from my preps. I perform RT-PCR on what I have which generates 25ul of cDNA and with a 1:5 dilution I do not have enough volume to run all my genes. Most of the genes generate a cycle threshold about 30-35 with 40 cycles. I'd like to be able to put more RNA in my RT reactions to lower the Cts a bit.
I took a look at the Statagene website and I believe that the kit is for generating cDNA from cells directly with out extracting the RNA first. (Or at least the kit I found did. Did I miss the one you were referring to?)
Epicenter (MessageBOOSTER cDNA Synthesis kit for qPCR) and NeuGEN (WT-Ovation Pico RNA Amplification System) both have RNA amplification kits available but they are very expensive, about $1500 for 10 reactions.
Do you have any other suggestions I could try?
-T

I had a look at the Stratagene info on their "Sidestep" products and it looks like you can purchase kits that do just the lysis & stabilization or kits that also include the mRNA purification and cDNA synthesis. The "SideStep QPCR cDNA Synthesis Kit" does do the cDNA synthesis directly from the stabilized cell lysate.

They report that with lysate from 17.5 HeLa cells, they can get a BAX gene detection CT at 36 cycles. With lysate from 175 cells this shifts to a threshhold crossing at cycle 32.



I understand the problem with getting detection of your genes so late on in the reaction (35-40 cycles). How about doing multiplex qPCR?

I usually just do 18S and my gene of interest, so just 2 different fluors but I think it is now possible to do at least 4 in the same reaction - if you have access to a qPCR machine that allows this multi channel detection.

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I saw an article in Nature Biotechnology a few months back which described the next generation of qPCR multiplexing. It allows thousands of different genes' expressions to be quantitated even more acurately than today's qPCR setups. for each gene of interest, a pair of primers is used. One primer is specific for the gene of interest whilst the other primer binds to the first primer and a special chip. I think it's this second primer which has the specific sequence tag (5 residues) that allows it to be distinguished from the other 16,000 permeatations. After mixing the RNA-containing sample with the primers and allowing these to interact with the chip, the chip is read, quantitating the number primer-bound transcripts that were present in the starting sample - no amplification required.

---------It sounds excellent but will take a while until it comes on the market.

I have been doing SYBRgreen so changing to multiplex would require that I change chemistries and start over with the primer design.

I'll take a look at the Stratagene site again.

Oh and did I mention that I am working in guinea pig and most of the genes that we are interested have not been published?

-T