SYBR® GreenER™ Two-Step qRT-PCR Kit for ABI PRISM® From Invitrogen

This kit is designed for use with real-time ABI instruments that are compatible with ROX reference dye at a final concentration of 500 nM. These instruments include the ABI PRISM® 7000, 7700, and 7900HT; the ABI 7300 Real-Time PCR System; and the ABI GeneAmp® 5700. This kit is not compatible with instruments that use ROX at a final concentration lower than 500 nM, including the ABI 7500. This kit consists of the two modules: the SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR module to generate first-strand cDNA from up to 1 ìg of purified total RNA, and the SYBR® GreenER™ qPCR SuperMix for ABI PRISM® module to perform qPCR.

To generate first-strand cDNA, you can use the SuperScript™ III First-Strand Synthesis SuperMix module. This module consists of 2X RT Reaction Mix [includes oligo(dT)20 (2.5 ìM), random hexamers (2.5 ng/ìl), 10 mM MgCl₂, and dNTPs] and RT Enzyme Mix [includes SuperScript™ III RT and RNaseOUT™]. In order to make a template, mix up to 1 µg RNA with 10 ìl of 2X RT Reaction Mix and add 2 ìl of RT Enzyme Mix, then add DEPC treated water to bring the total reaction volume to 20 µl. The manual suggests that the reaction incubates 10 min at 25°C, but I have found that elimination of this step in my system didn't affect the efficiency of the cDNA transcription and at times, even increased it. Perhaps, it could be because when I transfer RNA molecules directly from ice to elevated temperature, RNA molecules still stay non-coiled and provide better chance for random hexamers to bind to it.

The reaction is then incubated at 42°C to 50°C for 30-60 min. Other steps in the protocol suggest termination of the reaction at 85°C for 5 min and treatment of the reaction with 1 ìl (2 U) of E. coli RNase H, then incubation of it at 37°C for 20 minutes. I found that it was possible to omit these steps because 10 min at 95°C, which is the first step of real-time PCR, will terminate the reaction successfully. After cDNA transcription, the reaction may be stored at -20°C or used for real-time qPCR. cDNA can be used diluted or undiluted, but if diluted, the cDNA should be no more than 10% of the qPCR reaction volume (e.g., for a 50 ìl qPCR, use up to 5 ìl of undiluted cDNA). It is also possible to synthesize 1st strand cDNA by other methods for use with this kit. I successfully do it with Verso cDNA Kit (AB-1453/B), available from Thermo Fisher Scientific.

Following cDNA synthesis, use the reagents provided in the SYBR® GreenER™ qPCR SuperMix for ABI PRISM® module to perform qPCR. Prepare a master mix of common components, add the appropriate volume to each tube or plate well, and then add the unique reaction components (e.g., template). In the manual, the mix for a standard 50 ìl reaction is provided, but component volumes can be scaled as desired (e.g., scaled down to a 20 ìl reaction volume for 384-well plates, which also works for 96-well plates in the 7900HT ABI PRISM). The master mix should include the SYBR® GreenER™ qPCR SuperMix for ABI PRISM® diluted (1X) according your reaction volume, forward primer (200 nM), reverse primer (200 nM), cDNA template (max. 10% v/v). Place the reactions in a preheated thermal cycler programmed to perform a brief UDG incubation immediately followed by PCR amplification.

I use this kit routinely in my real-time RT-PCR experiments. In the course of my work, I test the expression of several genes in patients over a given period of time. Sometimes, I have needed to return back and test the expression of the gene in a control point or experimental sample several times. Using this kit, I almost always get consistent results, meaning that the Ct values of gene expression in repeated experiments are very similar to the first experiment.