I am very new to qRT-PCR, and have ran into a few problems that I don't know what to do
Every RT-PCR experts here left the lab, and I have no one I can ask for help, so any advise will be very much appreciates !!
1. The RNA quantity seems to be very low after the RNA extraction (bacteria sample) using QIAGEN mini RNA extraction kit, it's about 10 ng - 80 ng/ uL, is this concentration too low ? will I get a true cDNA synthesis from this low amount of RNA ?
I have tried to use everything new, started from new reagents, clean working space etc... but still get low quantity of RNA
2. the genes of interest have high GC content with about 66% - 70%, can I do qRT- PCR like how it is normally done? ie. reverse transcript at 48- 55 degree C, and amplify with SYBR green at 60 degree C? will the high GC content effect the result ?
The gene of interest I am analyzing is under expressed relative to the control where I expected to be over expressed, so I wonder if the high GC content will under estimate the amount of cDNA inputs ?
I have tried to do RT at 65 degree C, but then the Ct value of reference gene became much higher and unstable.
3. I use REST 2009 for calculating the relative expression level, and the output is giving as Log2 ratio. The genes of interest had very low level of expression compared to the control, eg. lower than log2 ratio of 1.0 or 2.0. what does this mean ?
Thank you so much for your help here!
Edited by Lisa Hsu, 04 February 2013 - 06:12 AM.