Concentration of cDNA after reverse transcription--no spec? - (Jun/29/2006 )
I"ve been reading the forum searching for a way to calculate the concentration of cDNA after reverse transcription. From what I've gathered, 1)I would need some instrument to help me quantify this, and 2)I would need to purify the cDNA. What I am really confused about is how my mentor managed to come up with a concentration with only the information listed here:
concentration of total RNA used
amount of total RNA used in the rxn
total volume of rxn mix
Can someone help me figure this out!?
Here's an example of the numbers:
concentration of total RNA 798 ug/ul
amount of total RNA used 6.27 ul
total volume of mix 20 ul (RNA, 1.73 ul water, 10 ul of cDNA synthesis mix)
Can someone PLEASE clarify!
From the Applied Biosystems book for quantitation of real time PCR:
"After RT, it is common not to quantify the resulting cDNA by UV absorbance. Instead the cDNA is assigned a concentration unit relative to the original concentration of RNA in the RT reaction. For example, if one loaded 10 μg of RNA into a 100 μL RT reaction, the designated concentration of the resulting cDNA would be 100 ng/μL; which means 1 μL of sample contains the cDNA generated from 100 ng of RNA."
I think that your RNA concentration is not logic ( so much for 1ul).
you must analyse your RNA on agarose to see if there is no dna contamination.
RNA can be cantified with UV spectometry 260nm.
if you use total RNA the cDNA amount is very low. I advice you to use column to purify your cDNA
I wont to know what your goal for the synthesis of cDNA (RT-PCR . cDNA library...)
i wait yiur reply