I am running real time pcr using probe method. My R square value is always 1, but my efficiency is between 70-85 only and my Ct for all samples are about 38.
I hope this article will help you
http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_053906.pdf
What about your positive control ????i mean the 18 s or any other endogenous control that you have. What is the ct value for that?
if you have a ct value of 38 that means there is negligible expression of your gene of interest. it could also mean that your primers are not correct. Did you check the melting curves. is there a single peak for your primer set?
Please provide more details, so that people can help you.
- pictures of the amplification curves (standards and samples),
- a picture of the calibration curve (dilution series),
- pictures of melting curves and gels of the product(s).
we need more information to be able to help you
especially about your positive control
R2 value indicates your prescision, how well you pipet (this is also aided if your reaction is optimised). So I read from this that your pipetting skills are good, but your reaction needs optimising and for us to help you, we need indeed more info.
What is the gene?, what are your reference genes, how did you do your std curve (1/2 serial dilution or logaritmic dilution?, meltcurve analyses on hte solo primer reaction since a probe needs a probe mastermix and does not contain any sybr green to visualise. What is your source DNA? Genomic or RNA converted into cDNA? How did you extract your RNA and how much RNA did you use to transform into cDNA, 1µg or less?, ....
A Cq of 38 is indeed way down, and not optimal at all since it introduces a lot of variability at that stage of the PCR. you need to get this between 15-25 to be ok, and for some HKG, that is not difficult, others are more difficult
@David: "R2 value indicates your prescision": no, surely not.
R² is a compound measure, look at its definition! For instance it strongly depends on the range of x-values (concentrations?) used. One may compare two R² values from the same setup (x-ranges), then the higher value indicates the higher precision, but this is a relative statement! Further, the R² depends on the distribution of values along the x-range. For instance, to take the extreme case: if you had only two values (at xmin and xmax), the R² will be exactly 1 (except y(xmin)==y(xmax)), so the R2 value is no measure of precision at all.
It is quite difficult to interpret R² (I never understand why this is such a beloved statistic shown in so many papers...), it is mostly misinterpreted, and it can only be interpreted in the precise context of the data/experiment/setup. An R² value given "in the outer space" is not at all informative, and to take it a if it would tell us something is usually the start of a series of misunderstandings and misconceptions.
Unless Sylvia does not tell us precisely how the R² value was obtained and from what data, an interpretation of the given R² value is simply impossible.
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