overexpression of a microRNA cluster - (Mar/06/2007 )
Hey fellow microRNA-ers!
I am wanting to create an artificial system in which a cluster of miRNAs is stably transfected into a cell line, which I will put into various oncogenic assays. The cluster is within one of 2 splice variants of a gene, with no known protein. So ideally I don't want to clone in the whole gene into a vector, just incase there are other factors in that gene that may play a phenotypic role.
How do I go about getting my cluster into the vector? How far up & downstream of the DNA sequence do I go to ensure I will get proper processing of all the members of this cluster? & do I need to clone it into 'normal' vector, or would an inducible system be more uselful? What kind of vector systems do people use for miRNAs? I've heard that Pol II or III are fine to express miRNAs, is this true? Also how do I monitor how effective my miRNA expression is? Im thinking TaqMan would probably be the best. I am new to all this, so any help that anybody could provide would be much apprectiated!
I guess this is a very hard question because we currently know little how microRNAs are transcribed and regulated especially you want to overexpress a cluster of microRNAs. If you clone the whole microRNA coding region into a vector and introduce it into cells, it is hard to predict how the primary transcrpt will get processed. Will it be processed the natural way? hard to say. Alternatively you can just cotransfect synthetic mature microRNAs for this cluster.
Hope these papers will help
Multiple shRNAs expressed by an inducible pol II promoter can knock down the expression of multiple target genes
Xu-Gang Xia, Hongxia Zhou, and Zuoshang Xu
BioTechniques Vol. 41, No. 1: pp 64-68 (July 2006)
Multi-miRNA hairpin method that improves gene knockdown efficiency and provides linked multi-gene knockdown
Daqian Sun, Margherita Melegari, Sunandini Sridhar, Charles E. Rogler, and Liang Zhu
BioTechniques Vol. 41, No. 1: pp 59-63 (July 2006)
Thanks for that - I will take a look at those papers!
I am going to screen the miRNA expression in tumours first by TaqMan to get an idea of which miRNAs appear to be the ones that stand out, or wether all of the cluster members are expressed to the same degree, & decide from there whether it will be better to transfect in those individual miRNAs that seem to be overexpressed, or just go ahead with the whole coding region, and verify by TaqMan that I am getting expression of the miRNAs in a similar way to what is happening in tumours. We shall see..
But I am having some issues with the AB miRNA TaqMan assays at the moment - I appear to be getting amplification of some of my miRNAs when I dont even put in any RNA in my RT reactions. It can't be a contamination in any of my RT reaction constituents since not all of them are giving me a consistent reading. It cant be a contamination in my PCR reagents since using water as my 'RT product' gives no reading. I am currently seeing if AB can shed some light on my issues, but has anybody else experienced any difficulties with their miRNA TaqMan kits..? I also noticed that the efficiency is not consistent on diluting my cDNA - as in a 1:10 dilution does not give a reduction of ~3 cycles to my Ct, but tails off rather too rapidly..all very strange!
Any advice will be mch appreciated!