especial cell line and gene, what is the result of RNA? - select a more inexpensive siRNA (Jan/30/2006 )
Hi. all and especially to Pcrman or fred_33:
I am a beginer in RNAi field and just want to use cell microinjection technique to study RNAi.
I also have another question to ask you. which is that, if the target gene of RNAi knockdown isn't expressed in cell culture, just in the brain of the living animal and at protein level, how can I test the expression of RNAi? And how can select a better siRNA from all candidate si RNAs more easily and more inexpensive?
max [font=Comic Sans Ms][size=7]
If the gene is not expressed at mRNA level in your cells, probably it is not transcribed. Why bother to knock it down by destroying its mRNA?
The cheapest way of finding a working siRNA target is to look in the literature.
Inexpensive ways of producing siRNA include in vitro transcription, siRNA casstte method. But I don't know if the potency of siRNAs such made is comparable to synthetic siRNAs.
if i understand, you want to set up an RNA assay against a protein you have in brain, but you don't have brain cell culture and the cells you have don't express it, in order to do an in vivo test?...
what i can recommend to you is the psi TEST from invivogen. link here
breifly, you clone the cDNA of interest downstrem the HTLV promoter and before cDNA of a secreted alkaline phosphatase. therefore cDNA is expressed and you can test siRNA. If siRNA rocks, you won't have the alkaline phosphatase in the media.
Then just pick up 00µl media and do a colorimetric test to estimate the efficiency of siRNA.