2x SensiMix dU SYBR® Green Kit From Quantace

The 2x SensiMix dU SYBR® Green Kit from Quantace is for use in real-time quantitative PCR (qPCR). The kit comes equipped with the reaction buffer which contains the heat activated DNA polymerase, dNTPs, dUTP, MgCl2, the internal reference and stabilizers. Quantace also provides the SYBR Green solution, extra MgCl2 and Uracil DNA Glycosylase (UNG). The kit has is available in 250, 500 or 2000 reaction sizes, when using a final volume of 50 ul per reaction. Primers specific for your gene of interest and your template are the only materials not provided.

I use the SensiMix kit for real-time PCR to quantify the abundance of messenger RNA in my samples using SYBR® Green. This is so that I can then quantify the relative expression of a particular gene at a particular time in my samples. I first convert the mRNA to cDNA using reverse transcriptase enzyme (not provided with SensiMix kit) and use that cDNA as template in the PCR reaction. Primers are designed as you would for standard PCR reactions and it is usually a good idea to test them using standard PCR too. This is because reagents used for semi quantitative PCR are cheaper and less precious then those used in real-time PCR. I usually design primers that are between 18 -22 base pairs in length and flank a region which has a sequence of between 100 – 600 base pairs.

The kit comes with a protocol sheet that recommends the volume and concentration to use for each reagent. They give the final volume of the PCR mix to be 50 µl; however, I usually do my reactions at a final volume of 10 µl and have had no problems. It is advisable to use a known concentration of cDNA as the template. I check for the expression of a particular gene between the control and target samples and then compare the expression of that gene to the expression of a housekeeping gene in both samples. (A housekeeping gene is a control gene that has a standard level of expression in all conditions.)

SYBR® Green is a double-stranded DNA-binding dye that gets incorporated into the DNA and fluoresces at 522 nm. More dye binds to the DNA as it is made and the fluorescence increases and is measured at each cycle; therefore, in ‘real time’. However it can also cause non-specific binding and it is important to design primers that don’t form dimers. I have had some problems when using the kit with highly diluted targets or when detecting genes that have a copy number that is much lower than that of the control gene.

The kit is designed so that it is also possible to use with fluorescent probe based reactions like TaqMan® probes and therefore SYBR® Green is not the only option. I have found the kit useful for the quantification of my genes of interest and I have been able to use it for both bacterial and human samples. Also since I use only 10 µl reactions, the kit lasts for a lot longer at -20°C and is more economically viable this way.