Reverse Transcriptase PCR - why radiolabeling? (Mar/20/2006 )
Hello everybody,
I hve just come aling a publication, where they do a RT-PCR (reverse transcriptase NOT Real time) and then blot the bands off the agarose gel onto a membrane. Using radioactive probes against the bands, they visualize them (looks like a northern). Why do they do this? Why don't they just show the gel? Is it for quantification purposes? 
-Jou-
hi
may you give the reference?
-fred_33-
Reference please!
-Matt
-MisticMatt-
Riccioni R, Pasquini L, Mariani G, Saulle E, Rossini A, Diverio D, Pelosi E, Vitale A, Chierichini A, Cedrone M, Foa R, Lo Coco F, Peschle C, Testa U. Related Articles, Links
Free Full Text TRAIL decoy receptors mediate resistance of acute myeloid leukemia cells to TRAIL.
Haematologica. 2005 May;90(5):612-24.
-Jou-
QUOTE (Jou @ Mar 20 2006, 09:16 AM)
Hello everybody,
I hve just come aling a publication, where they do a RT-PCR (reverse transcriptase NOT Real time) and then blot the bands off the agarose gel onto a membrane. Using radioactive probes against the bands, they visualize them (looks like a northern). Why do they do this? Why don't they just show the gel? Is it for quantification purposes?
I hve just come aling a publication, where they do a RT-PCR (reverse transcriptase NOT Real time) and then blot the bands off the agarose gel onto a membrane. Using radioactive probes against the bands, they visualize them (looks like a northern). Why do they do this? Why don't they just show the gel? Is it for quantification purposes?
Are you sure that was done after PCR?
Radiolabelling of the RT reaction was used to show that it has actually been produced cDNA.
That would normally be done by taking out a fraction of the RT-reaction and spiking with a radiolabelled nt. If the radiolabelled cDNA looked fine, the rest of the reaction would be used in a PCR reaction, or whatever the cDNA was made for.
-Gerd-