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Description
To complement the SensiFAST™ Probe and SYBR® kits, Bioline has developed the SensiFAST cDNA Synthesis Kit.
Product Highlights
Efficient – high-target affinity, coupled with a novel TransAmp buffer system for improved yield of full-length cDNA
Unbiased – optimized mix of random hexamers and anchored oligo dT primers for complete 5’ to 3’ RNA sequence representation
Sensitive – lower Ct values from a broad range of input cDNA concentrations, enabling accurate detection of very low-copy targets
Robust – reliable reverse transcription under challenging conditions, including complex templates and in the presence of inhibitors
Fast – high-yield reverse transcription from a very broad of targets in as little as 5 minutes
Product Description
The SensiFAST™ cDNA Synthesis Kit provides a rapid and sensitive method for first-strand cDNA synthesis, which displays excellent linearity across a wide range of starting material. This gives the same relative representation in cDNA templates, regardless of gene abundance, making it excellent for use in qPCR studies.
SensiFAST cDNA Synthesis Kit contains a highly-pure reverse transcriptase and optimized TransAmp™ buffer system, which includes a unique blend of random hexamers and anchored oligo (dT) primers to deliver the highest quality qPCR ready cDNA. This makes the SensiFAST cDNA Synthesis Kit ideal for working with limited sample volumes, such as laser-micro dissected samples and tissue biopsies (down to 1 pg of input RNA), to reverse transcribe precious RNA into stable cDNA ready for accurate real-time quantification. The unique blend of random hexamer primers and anchored oligo dT in the TransAmp Buffer also ensure unbiased 3’ and 5’ coverage and reverse transcription of all regions.
SensiFAST cDNA Synthesis Kit can be used with SensiFAST Probe and SensiFAST SYBR® Kits for fast real-time RT-qPCR without compromising on quality, giving real-time results in less than an hour.
Applications
Gene expression analysis
Tissue biopsy analysis
miRNA profiling / quantification
RNA target detection
Pathogen detection
Fig. 1 Speed and sensitivity
SensiFAST cDNA Synthesis Kit and a kit from supplier B were used for reverse transcription of a specific target from total RNA under fast conditions. The results illustrate that the SensiFAST cDNA Synthesis Kit (red) is more sensitive than the kit from supplier B (blue), as judged by the lower Ct values.
Fig. 2 Reproducibility of SensiFAST cDNA Synthesis Kit
SensiFAST cDNA Synthesis Kit was employed in 48 independent first-strand reactions, containing 100 ng total RNA and another 48 reactions containing 100 pg. The resultant first-strand product from each RT reaction was used in triplicate qPCR assays. The results demonstrate the excellent reproducibility of SensiFAST cDNA Synthesis Kit at high and low input RNA, as judged by the same Ct values.
Fig. 3 Unbiased representation across target genes
SensiFAST cDNA Synthesis Kit and a kit from supplier B were for reverse transcription of a specific target from total RNA. Primer pairs were then designed against the transcript at 1 kb intervals and used in qPCR reaction with SensiFAST SYBR® No-ROX. The consistent Ct values show that, unlike the results from supplier B (blue), the SensiFAST cDNA Synthesis Kit (red) did not show any bias across the target.
Fig. 4 Speed of SensiFAST cDNA Kit
SensiFAST cDNA Synthesis Kit was used for reverse transcription of a specific target from a 10-fold serial dilution of total RNA, for either 5 min (blue) or 60 min (red) at 42 °C. The results illustrate the high efficiency of reverse transcription over a broad linear dynamic range under both standard and fast conditions.
A discussion of the pros and cons of each detection strategy.
Our Institute consisting of more than 60 active researchers has a longstanding interest in understanding changes to the transcriptional landscape of prostate cancer. Reproducibility, sensitivity and cost-effectiveness are critical parameters in selecting our day-to-day cDNA synthesis kit. This combined with its convenient two reagent system continues to make the SensiFast cDNA synthesis kit stand out from its competitors and our preference for almost two years.
Martin Sadowski, Queensland University of Technology, Brisbane, Australia
Product Selection
Please refer to the Real-Time PCR Selection Chart to confirm the recommended qPCR mix for your real-time PCR application and instrument.
SensiFAST cDNA Synthesis Kit can be stored for up to 12 months at -20°C. Repeated freeze/thaw cycles should be avoided.
When stored under the recommended conditions and handled correctly, full activity of the kit is retained until the expiry date printed on the outer box label.
The SensiFAST cDNA Synthesis Kit has been designed for rapid generation and unbiased coverage of cDNA, especially for subsequent quantification with real-time PCR. The optimized 5x TransAmp buffer includes all necessary components for cDNA synthesis, including a unique blend of oligo dT and random hexamer primers and the RiboSafe RNase Inhibitor. The Tetro cDNA Synthesis Kit is the more flexible solution. You selects the desired setup, especially the desired RT primer. The Tetro Kit is optimized to work over a wide range of RNA concentrations and to obtain full length cDNA, which can be used for two-step PCR assays as well. Tetro is the recommended Kit for the generation of cDNA libraries and cloning.
For SensiFAST cDNA Synthesis Kit: The TransAmp Buffer already contains reverse transcriptase enhancers that reduce complex RNA secondary structure. For templates that have a high degree of structure, such as viral RNA and some plant RNA, we suggest using an additional 15 minute 48°C incubation step as mentioned in the manual. For Tetro cDNA Synthesis Kit: For samples with high secondary structure we suggest to denature the RNA template with primers prior to the reaction set-up. Furthermore, it is necessary to optimize the incubation time and temperature. The temperature of the RT step may be raised up to a maximum of 48°C.
The necessary amount of cDNA template for PCR or qPCR reactions may vary from the range of pg up to several hundred ng. It depends among other things on the analyzed target, the used application and sample characteristics. If you do not have empirical values for your assays please use the recommended amounts in the manuals of your PCR or qPCR assays and optimize if necessary.
It is not possible for the SensiFAST cDNA Synthesis Kit. Random Hexamer and oligo dT primer are already included in the optimized TransAmp buffer. It is possible to use gene specific primer for the Tetro cDNA Synthesis Kit to generate cDNA for specific target.
