Validating Microarray Data Using RT Real-Time PCR

This paper describes the use of real-time PCR for the confirmation of microarray data. Current publication guidelines require that all microarray results are confirmed by an independent gene expression profiling method. Real-time PCR is the method of choice for most researchers but it is not without drawbacks. The first step in confirming array results by real-time PCR is selection of gene-specific primer pairs. The process of design and optimization of gene-specific primer pairs can be a major bottleneck to array confirmation. With this in mind, SABiosciences has developed RT Primer SetsTM: pretested primer sets available for every human, mouse, and rat gene. RT Primer Sets allow you to quickly confirm any array result, removing the bottleneck of primer design and optimization and making them very cost effective. RT Primer Sets are designed for use with SYBR Green real-time PCR detection and can be used on almost any real-time PCR system. This paper shows how RT Primer Sets help research projects progress more rapidly and with less effort. A complete walk-through of an experiment using RT Primer Sets is provided along with discussion of considerations for successful real-time PCR. RT Primer SetsTM are a trademark of SABiosciences Corpoaration. SYBR Green® is a registered trademark of Molecular Probes, Inc. Applied Biosystems is a registered trademark of Applera Corporation iCycler is a registered trademark of Bio-Rad Laboratories 2 Validating Microarray Data Real-time PCR and SYBR Green Detection Real-time PCR monitors the amount of amplicon generated as the reaction occurs. Usually, the amount of product is directly related to the fluorescence of a reporter dye. Because it detects the amount of product as the reaction progresses, real-time PCR provides a wide linear dynamic range, demonstrates high sensitivity, and is very quantitative. The initial amount of template DNA is inversely proportional to a parameter measured for each reaction, the threshold cycle (Ct). SYBR Green-based detection is the least expensive and easiest method available for real-time PCR. Other detection methods (such as TaqMan chemistry) require expensive oligonucleotide probes labeled with fluorescent reporter dyes. Most real-time systems detect and accommodate SYBR Green making the method very flexible. SYBR Green specifically binds double-stranded DNA by intercalating between base pairs, and fluoresces only when bound to dsDNA. Detection of the fluorescent signal occurs during the PCR cycle at the end of either the annealing or extension steps when the greatest amount of double-stranded DNA product is present. However, SYBR Green detects any double-stranded DNA non-specifically. Therefore, PCRs using this detection method must generate single, gene-specific amplicons without the co-amplification of non-specific secondary products. RT2 Real-TimeTM PCR Primer Sets and Master Mixes Primers self-designed by researchers fail nearly 50 percent of the time, yielding non-specific amplification products and primer dimers. Pre-tested RT Primer Sets save significant time and effort. Primer sets are available for any gene in the human, mouse or rat genome in 24-reaction and 200-reaction scales. Each primer set is designed by an experimentally verified computer algorithm and then tested in a quality control assay to guarantee that they yield a single band of the predicted size by agarose gel electrophoresis. RT PCR Primer Sets and RT PCR Master Mixes combine to generate a complete assay optimized for SYBR Green-based detection on any real-time instrument. RT Real-TimeTM PCR Master Mixes are available with SYBR Green already added to the appropriate concentration for real-time PCR. Each is ready-to-use as a 2X solution. Instrument specific master mixes are available for Applied Biosystems real-time instruments as well as for the BioRad iCylcer. Each PCR master mix incorporates the reference dye required by the instrument manufacturer (ROX for ABI instruments, fluorescein for the BioRad iCylcer). A generic SYBR Green PCR master mix is also available without reference dye for use with other real-time instruments (such as the Cepheid SmartCycler). Protocol for Microarray Data Verification This protocol will describe how a result obtained by microarray analysis can be confirmed by realtime PCR. The microarray result used in this example shows that the expression of the human TNFAIP3 gene increases in HeLa cells upon treatment with TNFα (Figure 1). This experiment represents the simplest possible example involving one control sample, one experimental sample, and one gene-of-interest. More complicated analyses require a correspondingly more complicated experimental setup and protocol. For demonstration purposes, this protocol will describe the use of reagents available from SABiosciences including a complete reagent kit for reverse transcription, the ReactionReadyTM First Strand cDNA Synthesis Kit (Cat. No. C-01), as well as RT PCR Primer Sets and Master Mixes.