SunScript One Step RT-qPCR Kit
SunScript™ One Step RT-qPCR Kit is an easy and reliable system designed for fast RT-qPCR (real time) reactions based on detection of double-stranded DNA-binding dyes. One main advantage of the kit is the elevated temperature for performing the RT step thus allowing higher specificity of the reaction, and a higher likelihood in obtaining reaction products for difficult targets. Moreover, the excellent kinetic performance of the SunScript™ Reverse Transcriptase allows for speed optimization of the protocol.
The kit contains all the components needed to perform both reverse transcription and qPCR amplification in the same reaction by using gene-specific primers, in a “one step” reaction. This minimizes contaminations and allows higher reproducibility.
The system uses reaction mixes containing SunScript™ Reverse Transcriptase RNaseH+, a high quality HotStart Taq Polymerase, RNase inhibitor, and SYBR Green I, a double-stranded DNA-binding dye for a robust and effective quantification of RNA molecules. Intercalated SYBR Green I fluorophore can be detected using common settings for SYBR Green I (excitation wavelength ~450 nm, emission wavelength~ 520 nm).
SunScript™ Reverse Transcriptase RNaseH+ is an engineered version of the well characterized HIV-1 RT with increased thermostability. It minimizes RNA degradation for higher cDNA yields and allows to perform the reverse transcription step at temperatures up to 75ºC if needed (the recommended temperature for the RT reaction is 60ºC for most samples and depending on the melting temperature of the primers). These features make this kit the best choice for amplifying more difficult RNAs with a high degree of secondary structure or high GC content.
The reverse transcription reaction can be effectively achieved, while Taq
Polymerase remains inactive during this step due to blocking antibodies. The
inactivation/activation step at 95ºC provides an automatic “hot start” for the
Taq Polymerase to amplify the reverse transcribed cDNA.
The reaction mixes contain proprietary formulations which have been
optimized for both the reverse transcription and the qPCR reaction, including
dNTPs, MgCl2 and stabilizers (to provide a final concentration of 250 nM each
dNTP and 3.5 mM MgCl2 in the reaction).