Rep (4) : multiple m-RNA measurement
Mr SHIRE David
David.SHIRE at TLS1.elfsanofi.fr
Mon Feb 26 11:04:13 EST 1996
> > > lars.warngard (lars.warngard at astra.se.astra.com) wrote:
> > > : I am looking for a fast and easy method designed to measure the amount of different
> > > : (up to 10) specific m-RNA in the same run. I am glad for all suggestions.
There is no quick and easy way of measuring several specific mRNAs in the same run - detection, yes; absolute quantification, no; relative quantification can be fairly rapid. We and many others have developed multi-specific internal controls designed to measure mRNAs, particularly in the cytokine field where comparisons at the mRNA level can be very informative. Despite a lot of reticence on the part of referees, which is quite understandable, the method is slowly becoming accepted as a viable alternativ> e> to northern blotting, if one takes all the precautions necessary with RT-PCR.
It's true that designing and constructing the plasmids carrying the target sequences necessitates considerable skill and experience. In association with the European Cytokine Network editorial board, we at Sanofi Recherche have decided to distribute the plasmids we have available, together with a protocol, at absolutely no charge, simply your signature on a Supply Agreement - that is standard industrial practice. The idea behind this is that researchers in the field can get hold of tried and tested inter> n> al PCR controls, otherwise very difficult to get hold of, and share information concerning their use. Of course, the controls are not perfect, because they always lack a target that someone or other would like to measure and the housekeeping gene targets can be criticised. The latter is a great problem, often brought up in this Bionet discussion group and for which there is no easy solution. Just choose your own targets and insert them it into the plasmids by cassette mutagenesis or by PCR.
Furthermore, the European Cytokine Network journal has set up an Open Forum in which you can publish remarks, suggestions, improved protocols, new constructions for free distribution, etc. Our plasmids contain human cytokine targets (pQA-1 and pQB-3), mouse cytokines (pMus3 - see Science dated Feb. 16, 1996, pp. 987-990 for an example of its use) and a rat plasmid (pRat5) carrying a mixture of cytokines and neurotrophic factors (Mol. Brain Res. in press). In addition, Dr. Gutierrez-Ramos has announced in > t> he Open Forum that his plasmid carrying mouse cytokine receptor targets can be obtained on simple request.
So, Lars, if cytokine targets are of interest to you, just let me know and I'll give you as much information as I'm competent to do.
David Shire Ph.D.
Centre de Labege,
Labege-Innopole, voie no.1,
31676 Labege cedex,
Tel. (33) 61 00 40 54
Fax (33) 61 00 40 01
david.shire at tls1.elfsanofi.fr
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