High GC content RT-qPCR - please help - (Feb/04/2013 )
Dear all scientists,
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!
Hey, I hope I can provide some help.
1) Increase the suggested amount of bacteria for each RNA isolation. Decrease volume for RNA re-suspension. Your low abundance of RNA is not going to affect your RT-PCR, as long as you standardize each sample used. Check Qiagen's website and see what the relative concentration of RNA should be for a given bacterial cell number. Sometimes heating your RNA after resuspension (I use DEPC water, 65c for 10min) increases the RNA solubility, which will increase your concentration.
2) Every RT-PCR primer that our lab designs must fit a criteria. Mainly Tm, product size, GC content (listed in decreasing importance). Does the high GC content produce a Tm that would not allow you to use a 60c reaction with Sybr Green? Sybr Green has an optimal temperature, we usually never go above 58c (we have seen best amplification plots at this temp). How big is your gene? It is hard for me to believe that you cannot find at least two sites that will produce a decent Tm and GC %. If you need help, PM me and I will give it a shot.
Have you ran a gradient PCR with your primers and seen what the product looks like on a agarose gel? Look for a temp that gives sharpest band. Do the temps you are using now produce a smear?
Let me know if this helps.
the gene I am working with has a GC content of >66% and so is other genes in that operon.
I normally do standard PCR with annealing temperature at 67C or even 68C to give me a nice single band product.
This is why I am wondering whether doind qPCR at 58-60 C using sybr green would work and won't over estimate the Ct value? hence inefficient PCR?
Primer I use for RT-PCR has similar properties with the primers for reference genes.
Tm: 58-60C, GC%: 50-52%, Product size: 147-150 bp
Even the primers have GC% around 52%, but I checked the 148 bp product has a GC content of 66% !
do you think this will affect my RT-PCR result using sybr green?
It is a very good suggestion doing a gradient PCR with those primers.
I will give it a try tomorrow, and see how it goes on a gel
Thank you Jerry for you help here !
I do not think it will affect your Ct value. The Ct value corresponds to the cycling threshold, which measures the DNA synthesis when it exceeds the background signal. The GC content should not affect the incorporation of your Sybr green during DNA synthesis; therefore normal incorporation of Sybr green will occur. If you are worried you can use multiple constitutively expressed genes as a control and compare them to your high GC content gene.
Do a quick google search for "Sybr green high GC content gene," or something like that and find some literature that shows a RT-PCR rxn. May verify if they had any problems, but I doubt it.
Definitely do the gradient PCR and check and see what kind of product you get.