general inhibition of transcription - ways to check inhibtion of transcription (Nov/07/2005 )

Hi people
I was looking for a way to find out if my protein is inhibitin transcription. By means of RT-PCR we can see that the transcription of some genes is inhibited by this protein but what we want to know if this might not be a general phenomenon meaning that all de novo transcription is inhibited. we also looked at the transcription of actin and HPRT as controls and you can still find the RNA but this might be because these RNAs are quite stable and may thus not reflect active transcription of these proteins.
I hope someone out there has a helpful suggestion
stanley

hi
why don't you do an in vitro transcription assay? in my lab, a protein identified related with one of the factors involved in transcription shows significant inhibition when using nuclear extracts from cell surexpressing this protein versus normal-cells nuclear etracts

Fred, the invitro transcription might be a good idea, we are also looking into that. however, is a system that we do not have running at this lab. do you have a protocol that works on mammalian cells (HeLa and L929)?

something I've also heard about is a transcription run-off assay??? If I understood it correctly you isolate nuclei than the transcription which was taking place is 'frozen' you can than add radioactive labeled GTP and look at the incorporation???? this wil give you a measure of the ammount of transcription that was going on in the cell.
does anyone know a little bit more about this method? and how good is words?