ccubitt at codon.nih.gov
Tue Aug 1 09:08:04 EST 2000
What I have done in the past is use RT-PCR to amplify the pre-mRNA
transcripts from total RNA extracts. This can be accomplished using a
forward or reverse primer that binds to the pre-mRNA intron sequence and the
other paired primer that binds to another intron or exon. In this way the
relative transcription rate of the gene can be monitored. With more effort
this can be made semi-quantitative with standard quantitative RT-PCR
techniques. Below is a reference to the paper where I used this technique.
In this study I also found that actinomycin D shut down transcription within
30 min after which the degradation of the specific intronless mRNA could be
monitored by standard RT-PCR (a useful control). Hope this is helpful.
Differential induction of GRO alpha gene expression in human corneal
epithelial cells and keratocytes exposed to proinflammatory cytokines
Cubitt CL, Lausch RN, Oakes JE
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
38: (6) 1149-1158 MAY 1997
<aguacate2000 at my-deja.com> wrote in message
news:8m543e$odp$1 at nnrp1.deja.com...
> I am working with RNA from shrimp. I am actually
> trying to measure the half life of a messanger
> RNA. I read that one way to measure it is by the
> inhibition of RNA transcription, by actinomycin
> D. However, as I undesratand I have to measure
> the RNA synthesis rate in order to inhibit it.
> Somebody told me that I should do pulse labeling
> to know the synthesis rate.
> In the laboratory I am working, there is no
> chance to work with radioactivity.
> Is there a "normal" rate of RNA synthesis? Or in
> someway I could inyect to my shrimps certain
> amount of actinomycin to inhibit the
> transcription? (lets say mg of actinomycin per mg
> of tissue). My problem is the amount of
> actinomycin that I should inyect to my shrimp to
> be completely sure of the inhibition of
> Can someone give me a help???
> I really appreciate it.
> Sent via Deja.com http://www.deja.com/
> Before you buy.
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