High‐Throughput Real‐Time Quantitative Reverse Transcription PCR

Abstract

This unit describes the use of real-time quantitative PCR (QPCR) for high-throughput analysis of RNA expression. The topics covered include: design and validation of QPCR primers and probes for both SYBR Green-and TaqMan-based assays (see Support Protocol); the standard curve method (see Basic Protocol 1); an efficiency-corrected Ct (cycle time, also called cycle threshold or crossing point) method (see Basic Protocol 2); and the comparative cycle time, or Ct method (see Alternate Protocol). While the unit describes the use of the Applied Biosystems 7900HT (high-throughput, 384-well) instrument, the protocols may be utilized for any real-time PCR instrument. The highthroughput design allows analysis of the levels of transcript from a number of genes of interest (GOIs) at one time by using the appropriate primer set for each gene. (Within this unit, the term GOI will refer to the actual gene of interest as well as its RNA product or cDNA copy.)Because of the simplicity of the mathematical application, the standard curve method (Basic Protocol 1) is the most basic and straightforward QPCR assay described in the unit. In this method, standard curves are constructed for all of the GOIs from which RNA expression is being measured, and linear regression analysis is applied to interpolate arbitrary unknown sample values. The standard curve assay may be performed even if the PCR amplification efficiencies of the primer sets (as determined by the template dilution assay in the Support Protocol) are not equal, since correction for unequal efficiencies is intrinsic to the linear regression formula. One drawback of the standard curve method is that standard curves must be run for each of the primer sets on an assay plate. This results in less space on the plate for the unknown samples, and requires the use of additional reagents. This use of resources is particularly excessive when the PCR amplification efficiencies of the primer sets have been determined to be 100% and relative fold-change is the preferred outcome of the measurements. In such cases, the Ct method (see Alternate Protocol) should be employed instead. Another limitation is that unless the levels of all of the GOIs in the cDNAs used to construct the standard curves are known, the relative concentration of one GOI cannot be compared to that of another GOI. If comparison between the levels of different GOIs (without the knowledge of the relative level of the transcripts in the standards) is desired, the efficiency-corrected Ct method (see Basic Protocol 2) should be applied.The efficiency-corrected Ct method builds upon the standard curve method by incorporating PCR efficiency (E) into the quantity calculations. The standard curve slopes are used to calculate PCR efficiency according to the relationship E = 10 (-1/slope) . The efficiency has a maximum value of 2 for perfect doubling of the PCR template (see Basic Protocol 2 for an in-depth explanation of E). Note that in this method the standard curve is used only to determine slope and not to interpolate the RNA...

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