copies per ul to copies per gram tissue - (Jan/08/2014 )
I want to ask question about how to change your real-time pcr result.
I isolated RNA from, lets say 30 mg tissue (which the weight could be variable between one sample to another, but for example, just stick to the 30 mg). then I measured the concentration using nanodrop, and got the result 200 ng/ul (the RNA final volume is 30 ul). for the cdna synthesis, I took 100 ng total RNA, resulting in 20 ul cdna final volume, and for real-time pcr, I use 1 ul from the cdna with the final volume qpcr is 10 ul. then I ran the qpcr essay along with the standard curve, and assume all the reaction went well, I got the result 1000 copies RNA/ul.
The question is, how do I get to change the 1000 copies/ul into x copies/mg tissue? And if mathematical equation really can make it, is it r eally okay to change that? since it's involving molecular process, not only mathematical application?
thank you and sorry if you find my question a lil bit confusing.
It's relatively simple you used 100 ng cDNA in 20 ul therefore 100/20 = 5 ng/ul and used 1 ul or 5 ng (PCR volume should be irrelevant). This resulted in 1000 copies/5 ng or 200 copies per ng cDNA.
You got 200 ng/ul and 30 ul of RNA from 30 mg of tissue so 200 ng/ul *30ul=6000 ng total. 6000 ng *200 copies/ng =1200000 copies total from 30 mg.
Thank you bob1. Appreciate it :)