Technical Description Principles of Taq Man PCR

The use of the TaqMan reaction has been described in a number of original and review articles.[1,2] This approach makes use of the 5' exonuclease activity of the DNA polymerase (AmpliTaq GoldĀ®). Briefly, within the amplicon defined by a gene-specific PCR primer pair, an oligonucleotide probe labeled with two fluorescent dyes is created, designated as TaqMan probe. As long as the probe is intact, the emission of the reporter dye (i.e., 6-carboxy-fluorescein, FAM) at the 5' end is quenched by the second fluorescence dye (6-carboxy-tetramethyl-rhodamine, TAMRA) at the 3' end. During the extension phase of PCR, the polymerase cleaves the TaqMan probe, resulting in a release of reporter dye. The increasing amount of reporter dye emission is detected by an automated sequence detector combined with a special software (ABI Prism 7700 Sequence Detection System; Perkin-Elmer, Foster City, CA). The algorithm normalizes the reporter signal (Rn) to a passive reference. Next, the algorithm multiplies the standard deviation of the background Rn in the first few cycles (in most PCR systems, cycles 3-15, respectively) by a default factor of 10 to determine a threshold. The cycle at which this baseline level is exceeded is defined as threshold cycle (Ct) (Fig. 1). Ct has a linear relation with the logarithm of the initial template copy number. Its absolute value additionally depends on the efficiency of both DNA amplification and cleavage of the TaqMan probe. The Ct values of the samples are interpolated to an external reference curve constructed by plotting the relative or absolute amounts of a serial dilution of a known template vs. the corresponding Ct values.

In most cases of mRNA quantitation, gene expression has to be related to housekeeping genes that are expressed relatively stable throughout the cells. One major general difficulty in the use of housekeeping genes for the determination of mRNA transcript ratios is the possibility that their expression might also be altered by coexpression of pseudogenes and environmental changes (e.g., by hypoxia in case of glyceraldehyde-3-phosphate dehydro-genase). Pseudogenes may be eliminated using primer combinations that are intron spanning. However, unidentified influences cannot be dealt with as easily. Therefore, we suggest the use of at least two housekeeping genes for quantitation of mRNA expression. However, this aspect clearly needs further evaluation.

A different approach to quantify gene expression is the use of an external standard with a known gene expression or gene amplification. Although these preconditions are difficult to meet for mRNA quantitation, a reliable external standard can easily be obtained for N-MYC amplification at the DNA level because healthy volunteers usually do not carry more than two copies of the genomic sequence per cell in their peripheral blood.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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