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Description
Sensitive, reproducible detection of even the most challenging RNA targets, under fast thermal cycling conditions.
Product Highlights
Sensitive – optimized buffer formulation delivers reliable quantification from even very low copy number RNA targets
Reproducible – consistent results between technical replicates for increased confidence in results
Robust – reliable detection of RNA targets from a broad range of sample types
Fast – delivers reproducible, accurate assay results in as little as 40 minutes
Efficient – excellent performance in multiplex assays
Specific – antibody-mediated hot-start DNA polymerase minimizes non-specific amplification for improved assay sensitivity and reliability
Product Description
The SensiFAST Probe Lo-ROX One-Step Kit has been optimized for fast, efficient, unbiased cDNA synthesis and subsequent highly-sensitive, reproducible real-time PCR detection in a single tube. SensiFAST Probe One-Step has been optimized to deliver excellent results in both singleplex and multiplex assays.
An antibody-mediated hot-start DNA polymerase promotes rapid activation and supports highly-specific amplification, which in turn improves assay sensitivity and dynamic range. A combination of the latest advances in buffer chemistry and PCR enhancers confer superior assay performance under fast thermal cycling conditions. The inclusion of separate RiboSafe Inhibitor ensures accuracy by protecting RNA targets from RNase degradation.
The SensiFAST™ Probe Lo-ROX One-Step Kit has been validated on all commonly-used real-time instruments that require a low concentration of the passive reference dye ROX. SensiFAST Probe One-Step has been formulated for use with dual-labelled probes, including TaqMan®, Scorpions® and molecular beacon probes.
Mouse β-actin was amplified in triplicate using gene-specific primers and TaqMan probe according to each manufacturer's protocol, from 10-fold serial dilution of RNA, using either SensiFAST Probe One-Step Kit (red) or similar kits from Suppliers A (green) and Q (blue). The SensiFAST Probe One-Step Kit gives the lowest Ct values while maintaining reproducibility of technical replicates.
A fragment of the mouse β-actin gene was amplified in triplicate from a 10-fold serial dilution of mouse 3T3 total RNA using SensiFAST Probe One-Step Kit, gene-specific primers and a TaqMan probe, under fast thermal cycling conditions. The amplification plot shows excellent assay reproducibility.
A 100-fold dilution of human cDNA was targeted with 4 different gene-specific assays, in either singleplex reactions (dark coloured line) or quadruplex assays (light coloured line), using the SensiFAST Probe One-Step Kit and similar kits from Suppliers Q and T. The results demonstrate that SensiFAST Probe One-Step Kit is very efficient in multiplexing, as reactions containing single and four probes give the same Ct value, unlike the kits from Suppliers Q and T.
Introduction to SensiFAST
Overview, features and benefits of the SensiFAST product family
A discussion of the pros and cons of each detection strategy.
I compared the SensiFAST Probe One-Step Kit with another very popular product. SensiFAST Probe One-Step gave improved sensitivity, which was reflected in lower Ct values. Also, this product is a very good price. Value for money!!
Peony Fung, Cerberus Sciences, Thebarton, Australia
Instrument Compatibility
Please refer to the Real-Time PCR Selection Chart to confirm the SensiFAST Probe Lo-ROX One-Step Kit is compatible with your instrument.
BIO-78001: 100 x 20 µL Reactions: 1 x 1 mL BIO-78005: 500 x 20 µL Reactions: 5 x 1 mL
Storage & Stability
All kit components should be stored at -20°C upon receipt. When stored under the recommended conditions and handled correctly, full activity of the kit is retained until the expiry date on the outer box label.
Shipping conditions
SensiFAST Probe Lo-ROX One-Step Kit is shipped on dry/blue ice
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 recommended PCR conditions in the manual refer to the set-up of TaqMan probe-based PCR. If you want to use other probe types, it may be necessary to adapt the PCR setup conditions, please refer to the relevant literature. In case of hybridization probes it would be necessary to switch to a three-step PCR setup with annealing at 60°C and extension at 72°C and acquire the data at the end of the annealing phase (the exact annealing temperature may vary and has to be optimized for a specific assay). The two-step protocol with an annealing / extension step at 60°C would lead to the degradation of these probes, which is wanted for TaqMan but not for hybridization probes.
If you have further questions, please contact Meridian Technical Support.
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.
The amount of the ROX passive reference dye needed varies depending on the instrument optics. Our SensiFAST kits have been optimized for all these different instruments (see Product Selection Tool).
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.
