cq values for my reference coming like 22 and cq value of my target gene coming as 23 and 24 around?
can i considered this value for future calculation?
or what else i can do to get better cq value for my reference gene.
i diluted my sample 1:5 ratio. and i am using HPRT gene as a reference gene.
can somebody please guide me?
What's the problem with your Cq values, they seem pretty good. It is great to have Cqs around 20-30. Importantly, how stable is the expression of your reference gene HPRT. I'd rather have a Cq of 28 for a reference gene that is stable than have reference gene with a Cq of 22 but is unstable/less stable than the gene with 28 Cq (stability is important here). In your case, it just means that your reference gene is more abundant (2-4 fold) than your target gene. Your reference gene could be less or more abundant than your target gene, it matters whether it is stable or not (geNorm, RefFinder, BestKeeper etc).
There are softwares that will help you with reference gene stability. First, check to see if your PCR machine's analysis suite/software comes with that functionality. I know BioRad's CFX96 included this feature in their analysis suite. Then, you will need a panel of genes to conduct a reference gene stability test, that is you have about 4-6 genes (GAPDH, Beta-Actin, HPRT, etc) run at the same time (in the same run) for the experimental conditions of interest (so treated, untreated, diseased, non-diseased etc). I cannot emphasize the importance of all the conditions/parameters being included, never assume something is stable without testing it in that condition. I often see in post-docs when you ask them how they decided on their reference gene, they point out to a paper which sometimes has no clues whether the paper's author even conducted a reference gene stability in their own study, on top of that the post-doc's experimental conditions aren't exactly the same as the paper he/she was referring to. In my experience, it has been often the case that what works in 1 group doesn't work for another group, i.e. what worked for males doesn't work for the female group because the gene that was stable in males was not stable in females, and vice versa! Once you are clear on the reference genes, and your experimental parameters, you will need to decide which program/method to screen for reference gene(s). Again, here your real time PCR instrument may have this included in their analysis suite, but if it doesn't have a look into these ones: geNorm, RefFinder, BestKeeper & NormFinder. I have used geNorm before but that was via CFX96's analysis software that came with the instrument. They're all pretty good. Once you input your Cq/Ct values, they will have you an output value which you will then interpret to see whether one or more gene will suffice as a normalizer. Depending on the sample type, you may be able to use a single normalizer but sometimes with samples such as cancer cells you may need at least 2 reference genes to get reliable data.
Thank you so much for helping me and solving my problem beautifully.
I appreciate your answers and its worthy.
I have CFX OPUS 96 BIo Rad machine.
can you help me to set a protocol for the reference gene stability program into it?
if you have a protocol can you share it with me?
Which software does CFX Opus use, if you are using CFX Manager with Opus then I could help with that. BioRad's software is generally pretty user friendly and makes it easy to design plates.
Yes Kiessling I am using CFX Opus maestro the updated veriosn of manager.
but both are same.
you can guide me.
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