Standard Curves Real Time PCR

Standard Curves Real Time PCR

Standard Curves - Real Time PCR

Researchers can use several methods to quantitate alterations in mRNA levels using real time PCR. The standard curve method is an easy an basic method to use for real time PCR.

Copy number (for example, the number of copies of mRNA per nanolitre) and therefore mRNA abundance can be calculated from cells and biological samples using a a carefully constructed standard curve.

Amounts of RNA or DNA are then determined by comparing the results to a standard curve produced by RT-PCR of serial dilutions (e.g. undiluted, 1:4, 1:16, 1:64) of a known amount of RNA or DNA.

Housekeeping Gene Normalisation

To accurately quantify gene expression, the measured amount of RNA from the gene of interest is divided by the amount of RNA from a housekeeping gene measured in the same sample to normalize for possible variation in the amount and quality of RNA between different samples. This normalization permits accurate comparison of expression of the gene of interest between different samples, provided that the expression of the reference (housekeeping) gene used in the normalization is very similar across all the samples. Choosing a reference gene fulfilling this criterion is therefore of high importance, and often challenging, because only very few genes show equal levels of expression across a range of different conditions or tissues.

Common housekeeping genes used to normalize against:

• 18S rRNA
• actin
• GADPH
• RPLP0

How to Conduct a Standard Curve for Real Time PCR

To conduct a standard curve, a standard curve for any given primer set is produced by running a serial dilution of template. A plot is then made of the log(dilution/copy number) (x-axis) against the average CT value (y-axis).

Since PCR product accumulation is exponential, a halving of the starting template will result in a 1 CT value increase. If the priming efficiency is 100%, serial 2-fold dilutions of template will therefore produce serial increases of 1 CT value. It is more common to use a 10-fold dilution series which will produce serial increases of just over 3 CT units . Where this occurs the gradient of the line produced on a standard curve (log(dilution)/CT) value will be -3.2 .

The better the design parameters of the primers, the closer the standard curve gradient will be to -3.28. In practice, assays that have gradients -3.28 + or - 0.15 are good enough to be compared with other assays using the delta CT method of calculation. Compression of data at either end of the dilution series will flatten the line giving PCR efficiency above the theoretical maximum. This is most commonly seen where primer dimer formation has occurred. Steeper gradients than -3.28 show a decrease in the priming efficiency, either because one of the primers forms hair pin structures that interfere with annealing, or because the secondary structure of the amplicon interferes with primer binding. A further factor in priming efficiency is the overal lenght of the amplicon. The longer the amplicon, the lower the efficiency of amplification will be, and amplicons must be shorter than 150 base to ensure maximum efficiency.

Standard Curve Real Time PCR Protocol

The standard curve method can be used for relative quantitation where the internal control and target amplicons have different priming efficiencies. Overall it is less satisfactory than the delta CT method and should only be used when limitations of sequence or restrictions on the placement of primers prevent the design of assays compatible with Delta CT analysis. Instead of comparing the CT values directly, a standard curve is run for the internal control and target gene and this is used to calculate template concentrations of both for each data point. Normalisation is then archived by dividing the target signal by the internal control signal. In the example below, the 18S Ribosomal internal control amplifies at maximal efficiency (-3.24), but the target gene is less efficient (-3.75). The equation on this graph represent the mathematical formula to define a straight line (y=mX + C) where 'm' is the gradient of the line and 'C' is the intersecting point on the Y axis. If follows that from these standard curves the X value can be calculated for any y (CT) value by rearranging this equation. This must be performed separately for the internal control CT values and target gene values (Table 1). The normalised values are the relative expression levels of the target .

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