Hi I have a problem with optimizing TaqMan PCR? I am getting higher CT values in housekeeping gene GAPDH but undetermined target gene values. Also to be curious, I have ran those samples on 2% agarose gel and could not see any bands in target samples and only could see long smears in GAPDH samples, however template minus control was clear. I started cDNA synthesis with 2ug of cellular RNA with DNAse treatment.
- Unless your Ct cycles were < 25 you might see on an agarose gel as not sensitive enough
- Real time > 10 x as sensitive as agarose based stained products
- Your amount of starting RNA is good so I assume it is something about the quality of your RNA rather than quantity
- What are your 260/280 and 260/230 values ?
Hi,
how much cDNA are you using in the Reaction and to be specific how high is the volume of RT reation in your qPCR reaction setup? Reason is that RT reactions can inhibit PCR above a certain concentration
Thanks
Sven
- Unless your Ct cycles were < 25 you might see on an agarose gel as not sensitive enough
- Real time > 10 x as sensitive as agarose based stained products
- Your amount of starting RNA is good so I assume it is something about the quality of your RNA rather than quantity
- What are your 260/280 and 260/230 values ?
Hi,
The quality of the RNA material is important for successful cDNA synthesis. First, verify by taking OD
If the quality seems good, u can proceed for RT-PCR. I would like to know the probe systems.
You can improve ur target Ct value by increasing the primer and probe specific to the target or by reducing the primer and probe concentration of GAPDH. Either case, please do not make huge variation. For instance, if you have used 900nm set experiment with 800nm. Like this make very small adjustment. Make one change at a time.
Further, please verify the annealing temperature for your target, and GAPDH and the temperature you have used in the assay. Ensure that the detection/annealing temperature is optimal without compromising.
If thats good, give a TD-PCR approach for few cycles with higher annealing temperature or temperature closer to the target. so that the target will be amplified little earlier. Note: This method is often suitable for qualitative assays. However you can try to figure out the nature of your experiment.
all the best
Sven Reister Thanks for your reply. I used 2ul of 1:5 diluted cDNA in 20ul taqman reaction.
Laurence Stuart Dawkins-Hall thanks for your reply. My ODs with nanodrop at 260/280 were between 1.86 to 1.95. I used 2ul of 1:5 diluted cDNA in 20ul taqman reaction.
Narayanan Kalyanaraman thanks for your reply, I used TaqMan probe system that come with predefined annealing temperature as you know we just need to feed the serial number in qPCR instrument which automatically detects TMs of probes.
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