For SYBR® Green real-time one-step RT-PCR analysis and probe-based real-time one-step RT-PCR and multiplex RT-PCR analysis from cultured cells without RNA purification

Features

No RNA purification required, significantly saving time

Just 3 steps from cells to real-time RT-PCR

High-throughput analysis of 96- and 384-well plates

Unique gDNA Wipeout Buffer enables detection of RNA only

Immediate startup using optimized reagents and protocols

Product Details

FastLane Cell RT-PCR Kits accelerate and streamline real-time RT-PCR and are ideal for experiments requiring rapid high-throughput gene expression analysis. The kits provide a set of buffers for preparing FastLane lysates from cultured cells. In addition to lysing cells, the buffers also stabilize cellular RNA and eliminate genomic DNA. FastLane lysates are used directly in real-time one-step RT-PCR and multiplex, real-time one-step RT-PCR. A variety of detection formats are available, including singleplex detection with SYBR Green or sequence-specific probes or multiplex detection with sequence-specific probes (with or without ROX passive reference dye).

Performance

The FastLane Cell SYBR Green Kit uses a unique RT-PCR buffer to prevent the formation of nonspecific products and primer–dimers, allowing highly specific quantification in real-time one-step RT-PCR. Sensitive quantification is also achieved despite the absence of an RNA purification step . Results in high-throughput experiments are very reproducible  and are not affected by genomic DNA contamination.

The FastLane Cell Probe Kit delivers sensitive probe-based detection in real-time one-step RT-PCR without the need for RNA purification. Real-time quantification is more sensitive and linear compared with similar kits , and is highly reproducible . In addition, genomic DNA does not interfere with quantification.

With FastLane Cell Multiplex Kits, multiplex analysis in 96- and 384-well formats is highly reproducible . The kits are ideal for rapid, high-throughput gene expression analysis, such as validation of siRNA-mediated gene knockdown . Target and control genes are amplified in the same reaction with similar, high PCR efficiencies, enabling relative quantification by the ΔΔCt method.

Principle

FastLane Cell RT-PCR Kits use FastLane technology to prepare cell lysates for real-time one-step RT-PCR and multiplex, real-time one-step RT-PCR. The kits accelerate and streamline real-time PCR analysis of cultured cells by eliminating the need for RNA purification and deliver reproducible results in high-throughput analysis, enabling easy processing of 96 or 384-well plates.

With the FastLane Cell SYBR Green Kit, specific transcript detection is guaranteed in real-time PCR analysis of cells when the kit is used in combination with QuantiTect Primer Assays. These are predesigned, genomewide primer sets, available at GeneGlobe.

All types of probe chemistry are compatible with the FastLane Cell Probe Kit, including hydrolysis probes (e.g., TaqMan® or other dual-labeled probes), LightCycler hybridization (FRET) probes and Molecular Beacons. A ready-to-use RT-PCR master mix containing a novel RT-PCR buffer and a hot-start DNA polymerase eliminates the need to optimize probe concentration and other PCR parameters.

FastLane Cell Multiplex Kits are ideal for rapid, high-throughput gene expression analysis, such as validation of siRNA-mediated gene knockdown. Unlike other methods, FastLane Cell Multiplex Kits deliver success in multiplex, real-time one-step RT-PCR at the first attempt due to an optimized master mix containing a novel RT-PCR buffer and a hot-start DNA polymerase. Two kit formats are available: the FastLane Cell Multiplex Kit for cyclers that require ROX dye for fluorescence normalization, and the FastLane Cell Multiplex NR Kit for all other cyclers.

Procedure

With FastLane Cell RT-PCR Kits, lysates are obtained in just 12 minutes in a 3-step procedure: cell wash; cell lysis with integrated RNA stabilization and genomic DNA elimination; and lysate pretreatment. After pretreatment, lysates are ready to use in real-time one-step RT-PCR and multiplex, real-time one-step RT-PCR.

The kits contain sufficient reagents for cell lysis in two 96-well cell culture plates and subsequent real-time RT-PCR and multiplex RT-PCR (50 µl PCR volume). The kits can also be used to analyze a single 384-well cell culture plate if the PCR volume is reduced to 20 µl; if a 50 µl PCR volume is desired, an additional QuantiTect SYBR Green RT-PCR Kit (200), QuantiTect Probe RT-PCR Kit (200), QuantiTect Multiplex RT-PCR Kit (200) or QuantiTect Multiplex RT-PCR NR Kit (200) needs to be purchased.

Applications

FastLane Cell RT-PCR Kits are ideal for experiments that require rapid, high-throughput gene expression analysis, such as:

Validation of siRNA-mediated gene knockdown

Screening of potential drugs

Detection of stimulated gene regulation