Detection of mi/shRNA - (Feb/07/2008 )

Hello,

I have a question: I created cell lines expression shrna and mirna constructs for gene knockdown and now I want is to detect if the sh/mirnas are expressed. I don't need fancy real-time /Taqman stuff to find out about expression level or similar, just: is it there or not?, just a cheap system to detect my introduced sh/mirnas. I don't have enough cells for northern blot and no equipment for radioactive work. Is there a simple and cheap rt-pcr method without sybr green or taqman?

Thank, stardust

Hi there,

I have a reporter gene and i can even see a knockdown of my target gene but I have to prove that my sequences are expressed...

Stardust

you may consider the possibility of the psiTEST system. I used it during my thesis and it's a nice indirect way to see if shRNA expression occurs in your cells.

Hi Fred,

I have already done this as well...but as you said it is only a indirect detection method...i would need a simple direct method...does anyone know a simple sybr green base pcr assay for it?

Stardust

I don't think that a SYBR qPCR assay would work because of the short size of the RNA (this is why hairpin primers are used for TaqMan miRNA). Maybe not simple, but could an in situ hybridization on your cells work? There is also an ISH in solution kit from Ambion (I haven't used it though).

which kit from ambion do you mean?

I would recommend just a regular rt-PCR (not real-time). you would create primers to a pre-miRNA, it will not give you the info on mature miRNA abundance. however most of papers published to date show that pre-miRNA are a good indicator of mature miRNA abundance. and regular PCR is SO MUCH cheaper. you would assay your results using 4% agarose gel. it will give you estimate how your sample compares to the control. remember to include internal calibration control --- e.g 5S or U6.