Hi!

I would recommend in vitro transcription over exponential amplification.. For sure, you can never expect that the amplification is totally unbiased but at least, or as far as I have read so far, the results are more representative and reproducible. On the other hand with the kits available around you can get incredible amounts of RNA.

What kind of sample do you have? Is it prokaryotic or eukaryotic? Are you amplifying total or mRNA?

Hi,

Thx for comment. Actually I'm thinking of disseminated human tumor cells in peripheral blood circulation. I'm completly new in this field as my expertise is on single cell DNA amplification.

Besides from being a beginner further difficulties may be the fact that I need to work on labeled cells and that I can't really get rid of the genomic DNA (due to protocol/technical reasons).

Back to your questions:

Eukaryotic mRNA

Hopeless?

Hi Thomas,

Ahaha, no, don't worry, it is definitely not hopeless. I just can't understand why you are not able to get rid of genomic DNA.... Anyway, to me that shouldn't be that big a deal. Check this out:

http://www.ambion.com/catalog/CatNum.php?1751

What you basically do is to incorporate a t7 promoter region to your RNA during reverse transcription, and than do ds cDNA synthesis. afterward you ligate the total RNA into a vector and the T7 RNA pol. translates this cDNA into a lot of RNA. it says starting from 100 ng but I have used it down to 10, and you still get magnificent amounts. Since gDNA does not have the promoter region, it won't be amplified -though might still remain in some amount-

What worries me is the labeling.... how are the cells labeled? Or even more important, how were they fixed before labeling? That can have a huge impact on the RNA -especially the mRNA- quality. And how are you planning to extract the RNA? I think with this kit you cannot start from single cells without extraction... then we can think of sth else.

Hope this was helpful!

Hi,

The plattform I'd like to use is a slide capable of PCR (AmpliGrid). I use this slide (surface modified) to collect my cells after laser microdissection. Once the cells are on the reaction area of this slide one can do PCR in about 2µl. This works with single cells and whole genome PCR and, well, DNA.

I thought of adapting this low-volume approach to expression analysis.

Drawback of this technology is the need to stay in one batch. There's no chance of clean-up. The only way to go ahead in the protocol is to add reagents to existing volumes.

For PCR it works like this:

Cell lysis in 0.75µl lysis mix (covered by 5µl sealing oil)

After lysis you add 1µl of PCR mix (I do 16plex-PCR/DNA fingerprinting) to the 0.75µl of lysed sample and do PCR (on a slide cycler).

I once did RT-PCT of human GAPDH of single (unfixed) cells and it worked (was kind of a surprise to me).

So I thought of doing a whole transcriptome assay ...

-Cells will be IF-labeled and counterstained with, lets say, DAPI

-For DNA analysis I fix the cells in PFA (2min)

-NO EXTRACTION

Haven't checked for the kit yet (this project will start of in spring at earliest) but if theres no way around RNA-DNA separation this protocol already died ...

Good Morning,

That seems a bit tricky indeed... You have to find a way of selecting for mRNA, otherwise for sure you\'ll never know if your signals are from the genome or transcriptome..

Hhmm, first of all, I think the fixation for 2 min is not that big a deal for the mRNA, at least the same procedure is also being used for mRNA FISH... but I have no experience if DAPI staining is bad for the RNA or not, I wouldn\'t think so but I would first try to find literature or do some incubation experiment to see if the RNA is degrading with DAPI staining.

Regarding to the single cell mRNA amplification, I have a Nature paper somewhere in my archive, it is Nature Biotechnology 20, 387 - 392 (2002) doi:10.1038/nbt0402-387... Perhaps you also know it, they do magnetic separation of mRNA with paramagnetic oligodT and proceeding with PCR... which means to me that you can still stick with the same principle till the cell lysis stage...

I would take a look at the paper. But if you want to go for the mRNA, you have to do a separation at some stage, I think there's no way around it. I will still think about it.

Hi Basak,

Thx for keeping an eye on that, and for the lit. I will have to rest this idea for some a time...

:-)

Ah, no worries. Let me know what comes out of it:))