"cDNA must accurately represent RNA input amounts" - (Jun/13/2005 )
The protocol: "The RT step is critical for sensitive and accurate quantification and the amount of cDNA produced by the reverse transcriptase must accurately represent RNA input amounts. "
What does it mean that “the amount of cDNA produced by the reverse transcriptase must accurately represent RNA input amounts” and how can we see it?
-indoubt-
QUOTE (indoubt @ Jun 13 2005, 10:02 AM)
The protocol: "The RT step is critical for sensitive and accurate quantification and the amount of cDNA produced by the reverse transcriptase must accurately represent RNA input amounts. "
What does it mean that “the amount of cDNA produced by the reverse transcriptase must accurately represent RNA input amounts” and how can we see it?
What does it mean that “the amount of cDNA produced by the reverse transcriptase must accurately represent RNA input amounts” and how can we see it?
It means regardless of what RT-primer set you use (random hexamers, DT-Oligos or GSP), you will want to have full transcription of the RNA you are targeting.
Any commercial RT-enzyme you use, usually comes with a protocol that gives specification of how much enzyme and primers should be used to get good transcription.
Also, you could use Rnase H incubation after the RT step and then use a spec to see how much cDNA you have and compare that to starting amount of rna you used.
-dobbiewalton-