Real-Time Polymerase Chain Reaction and Melting Curve Analysis - Clinical Applications of PCR - page 24

3.4. Temperature Cycling for Real-Time PCR and Melting Analysis
Place the sample carousel into the LightCycler and program the desired
temperature conditions for PCR ( see Note 9 ). Begin thermal cycling and
acquire fluorescence each cycle at the end of the extension phase. If melting
will be performed in the LightCycler, the melting conditions are also entered
before amplification. If a high-resolution melting instrument is available ( see
Note 10 ), the capillaries are transferred after PCR from the LightCycler to the
high resolution instrument for melting.
3.5. Data Analysis
For absolute quantification, standards that bracket the potential concentra-
tion range are run in parallel. Typically, four to seven standards are separated
in concentration by factors of 10. Replicates are not necessary, unless the
instrument or preparation method are compromised or very low copy numbers
(<20 copies per reaction) are being analyzed. A negative control (no template)
should also be included in order to identify any background signal, although it
is not used in quantitative analysis. Amplification plots or “growth curves” are
displayed by plotting fluorescence vs cycle number ( Fig. 2 ). A quantitative
standard curve is constructed by relating the log of the initial number of
templates in each standard to a fractional cycle number derived from each
curve. Although a threshold fluorescence level can be used to define these
fractional cycle numbers, the second derivative maximum is more precise and
depends on the shape of the curve rather than on a specific fluorescence value
( 2 ) . Unknown concentrations are determined by interpolation from the stan-
dard curves.
For relative quantification, absolute standards are not necessary. The rela-
tive concentration of target between an experimental and a control sample
depends on their fractional cycle numbers and the PCR efficiency ( Fig. 3 ). For
well optimized PCR reactions, the efficiency is close to 2.0 and the relative
concentration depends only on the fractional cycle numbers. When greater
accuracy is required, the PCR efficiency can be derived from the standard curve
slope of template dilutions. In some experimental designs, the overall amount
of nucleic acid (e.g., mRNA) may be difficult to control. In this case, it is
common to normalize the test gene against a reference gene, such as a “house-
keeping gene” that is assumed not to vary with the experimental conditions.
Melting analysis with SYBR Green I is commonly used to characterize PCR
products ( 3 ) . A recent development is high-resolution melting analysis,
enabling even single-base genotyping within PCR products more than 500 bp
in length ( Fig. 4 ). Samples are amplified in the presence of the dye LCGreen
and melted at 0.3°C/s with 50-100 data points collected per degree Celsius.