Hi Everyone,
I'm reaching out for some guidance regarding conducting a comparative Ct analysis with the ABI StepOnePlus system, and I hope you can help shed some light on my dilemma.
Here's the situation: I'm working with 85 samples arranged in a 96-well plate configuration. Given that I have around 14 genes to analyze, and a housekeeping gene like GAPDH, I'm encountering some logistical challenges.
With a 96-well plate, I can allocate 12 samples per run for 7 target genes and a GAPDH as housekeeping gene. Then, for the next batch, I repeat the process with the remaining genes. However, this means that GAPDH will need to be re-run for the same 12 samples, and considering my limited materials, this poses a significant constraint.
Therefore, I'm considering the option of running the reference gene (e.g., GAPDH) separately from the target genes. This way, I can optimize sample allocation and make the most of my resources.
It's worth noting that I only plan to extract the Ct values and perform delta-delta Ct analyses independently.
My primary concern is whether this approach might introduce inaccuracies or issues in the delta-delta Ct analysis process, especially considering the potential variation between runs.
Your insights and experiences on this matter would be immensely valuable to me!
Thanks in advance for your help
I've had a similar situation when I was running 96-well CFX96 plates.
What we did was to keep the reference + target genes on the plate for the samples used, but split the samples across 2 runs. I had total of 6 samples and 4 groups run in triplicates, so we ended up using 3 samples per plate which had the reference and target gene on plate 1 for samples 1-3, then reference and target gene on plate 2 for samples 4-6. In that scenario, you will need a calibrator sample to keep track of the Ct drift across the runs, your calibrator sample can just be a mixture of the cDNA of some your samples. Some software may help you with this, I know CFX96 software does this for you. The software will then merge the different run(s) and give you a delta Ct for the different batches. I cannot predict how favorable the Ct drifts will be across your different runs for you. Looking at your sample numbers and genes count, would it be easier to just run some targeted NGS panels? There are service providers in Turkey who has NGS services that might be able to provide an NGS solution depending on the budget availability.
you need to use a plate control to ensure plates are run the same. choose one sample with one set of primers and include it on all plates (in triplicate or quadruplicate). normalize to the plate control then your reference gene.
it does not have to be a experimental sample for gene it can be from untreated cells for any gene. you must prep the qPCR master mix for the plate control all at once in a big batch and use it on all plates.
just a little insight for you: if you plan to run the experiment in two separate plates, do prepare both plates at the same time. While one plate is running, the other is kept on ice or in the fridge, protected from light. Then, after the run of both plates, you will need to obtain the threshold line that gets you your Ct values manually so that you can adjust it to be the same for both runs. I think you still need to consider also the insight shared by Julie in order to really make sure the outcomes of both plates are really the same even if you already adjust the threshold line to be the exact same one.