hi all,
i've decided to use RNAi as a method of functionally studying my gene of interest, and have a qeury you guys might be able to help me with.
in the absence of a fluorescent or confocal microscope, how can i confirm the gene silencing effect before looking at functional experiments? can i use basic RT-PCR or do i have to use some other method?
any help is greatly appreciated
james
-flashboy-
i am doing experiments about gene silencing. i think you should get the antibody against the gene of your interest, because the immunohistochemstry or western blot is easy to detect whether your gene silencing is successful. may your experiments success!!
-lwk2003-
thankyou very much, i already have the antibody so i think i'll try westerns...... i hope yours go well too!
-flashboy-
you can also try a northern blot or real-time PCR
-ros-
if only we had the money to buy a real-time pcr cycller..... ah well.
-flashboy-
RNA silencing has two major routs. At the transcriptional level and at the post-transcriptional level. this is at the molecular level. If you have a phenotype of your gene, then the best is to see if the phenotype is mimiced in your RNA silencing experiment. At the molecular level, RT-PCR using gene specific primers will work. Quantitative RT-PCR is comparable to real time PCR.
Good luck.
In my case, my gene gives no visible phenotype and its knockout function give no phenotype either. thus my advise is to seek a phenotype to assess the functional importance of your gene.
-is52mih-
hi ,i am doing RNAi now ,I have the same query : if I want to monitor the effenciency of RNAi by semi-quantitative RT_PCR , how to conduct it ?I mean ,how to determine the amout of cDNA or RNA as templete ,and how to determine the cycle number ,it is limited to log phase? I have tried ,but the result is varied when change the cycle number and the templete amout!
-dingdang-
Basically I would use RT-PCR (conventional or real-time) to compare transcript levels from a control to transcript levels from an experimental. I suppose you could do a Northern blot as well, but it would be more involved. At the very least this will tell you if you're transcript of interest is being knocked down.
-Hank
-haringsh-
