SensiFAST^™ SYBR^® Lo-ROX Kit

Fluorescein is an alternative passive reference dye used just in the Bio-Rad instruments (see Product Selection Tool), the SensiFAST SYBR & Fluorescein contains fluorescein premixed in the mastermix at optimised concentrations.

For your convenience the SensiFAST kits have an optimized amount of SYBR® Green in the mastermix.

This is not possible because the SYBR® is pre-mixed into the SensiFAST SYBR® mastermix.

SensiFAST One-Step Kits can be used with most RNA/cDNA templates. To give you some idea of a few of the types of templates used with these kits: Human - Rosato R.R., et al. Cancer Res. 67: 9490-500 (2007) Rat - Remund K., et al. Expt. Lung Res. 35: 359-370 (2009) Drosophila - Brown A.E., et al. PloS ONE 4: e4490 (2009) Nitrogen-fixing bacteria - Bahlawane C., et al. Mol. Plant-Microbe Inter. 21: 1498-1509 (2008) Diphtheria bacteria - Jochmann N., et al. Microbiology 155: 1459-1477 (2009) Green algae - Wobbe L., et al. PNAS 106: 13290-13295 (2009) Bovine virus - Park S-I., et al. J. Virol. Methods. 159: 64-6 (2009) For cDNA/DNA templates SensiFAST SYBR® and Probe kits are used. To give you some idea of a few of the types of templates used with these kits: Stem Cell - Bernardo A.S., et al. Stem Cells 27(2): 341 -351 (2008) Cow - Baumert A., et al. J. Dairy Research 76(3): 356-364 (2009) Mouse - Hoyles R.K., et al. Arthritis Rheum. 58(4): 1175-88 (2008) Quail - De Winter P., et al. British Poultry Science 49(5): 566 – 573 (2008) Insect - Bass C., et al. Malaria journal 6 111 (2007) Fish - Miller M.R., et al. J. Nutr. 138(11): 2179-2185 (2008) Crab - Wilcockson D.C. & Webster S.G. Gen. & Comp. Endo. 156: 113–125 (2008) Nematode - Murray S.L., et al. Mol. Plant-Microbe Interactions 20(11): 1431–1438 (2007) Plant - Hecht V., et al. Plant Physiology Preview DOI:10.1104/pp.107.096818 (2007) Yeast - Kawauchi J., et al. Genes & Dev. 22: 1082-1092 (2008) E.coli Bacteria - Saeed H.A., et al. Research J. Microbiology 4(4); 173-177 (2009) Virus - Muscillo M., et al. Water, Air, & Soil Pollution 191: 1-4 (2008)

Although SensiFAST kits have been designed for fast PCR on the new generation of fast machines, they will work equally well for standard or fast PCR protocols on all real-time PCR machines.

Polymerase activity during the reaction set-up causes non-specific amplification including primer-dimer formation. To avoid non-specific amplification the polymerase is only activated after heating at 95°C for 2-3 minutes.

When comparing SensiFAST with a mix from another supplier we strongly recommend to use the suggested conditions for PCR setup and cycling. Every real-time mix has its specific composition and different properties. That’s why they should be tested with their specific conditions, mostly this is possible on different plates only. We recommend amplifying from a ten-fold template dilution series. Loss of detection at low template concentration is the only direct measurement of sensitivity. An early Ct value is not an indication of good sensitivity, but rather an indication of speed.

The emission recorded from ROX during the baseline cycles is used to normalize the emission recorded from the reporter (SYBR®) in later cycles in some instruments. ROX compensates for small fluorescent fluctuations such as bubbles and well-to-well variations that may occur.

Why are the ROX concentrations different?

What could be done to avoid unspecific products in melting curve analysis?

SYBR® Green I is an inexpensive, universal dye which binds to all dsDNA. It can be easily used in combination with a simple primer pair to detect PCR products in real-time. This dye is very attractive for researchers analysing lots of different genes.

The probe system is always specific, probes only detect the gene of interest. It is also possible to distinguish between similar sequences with small differences like SNPs or mutations. In general, probe assays need less optimization than SYBR® Green I assays.

This method is only effective if all the researchers in the laboratory, or using the thermocycler, are all using a dUTP/dNTP and UNG system. If even one researcher is not, it ceases to be an effective control. Using dUTP has also been shown to be inefficient as it increases the Ct values by reducing reaction efficiency. Most labs do not need to use the dUTP method of control, optimised protocols will allow high specificity of PCR and good lab practices (using disposable consumables, the use of filter tips and maintaining a separate area for PCR set-up and PCR amplification and any post-PCR analysis) virtually eliminate the risk of cross contamination.

In a one-step reaction the reverse transcription reaction and the real-time PCR reaction are done in one tube, making this a closed tube assay, so contamination can be avoided. It saves pipetting steps and time and is easy in handling, making it ideal for high throughput screening. In a two-step reaction the reverse transcription reaction and the real-time PCR reaction are done in separate tubes. It gives a more flexible way of working in that the cDNA can be used for more than one real-time PCR reaction and can be archived, eliminating the need to continually isolate RNA. For convenience Meridian sells the SensiFAST cDNA Synthesis Kit separately if you wish to take a two-step approach.

A possible reason is that the template amount is too concentrated. An early Ct may impair the baseline correction and may cause these strange curves. We suggest to dilute the template. Alternatively if the curve is flattened, this may be due to inhibitors in the reaction. Running a serial dilution of your sample will dilute the inhibitor concentration, if this corrects the problem, you will either need to go back and re-purify your template, or use it at a lower concentration, where the inhibitor effect has been diluted out.