For probe-based real-time one-step RT-PCR analysis from cultured cells without RNA purification

  • 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

The FastLane Cell Probe Kit accelerates and streamlines real-time RT-PCR and is ideal for experiments requiring rapid high-throughput gene expression analysis.The kit provides 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 probe-based real-time one-step RT-PCR. A variety of other detection formats are also available, including detection with SYBR Green or multiplex detection with sequence-specific probes (with or without ROX passive reference dye).

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FastLane Cell Probe Kit (200)
FastLane Cell One-Step Buffer Set, 2x QuantiTect Probe RT-PCR Master Mix, and QuantiTect RT Mix
216413
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The FastLane Cell Probe Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

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Sensitive probe detection without RNA purification.|Greater sensitivity than other kits.|Highly reproducible analysis of a 384-well plate.|Linear detection down to a single cell.|
Expression of β-actin (cytoskeleton protein) was analyzed by real-time one-step RT-PCR. The FastLane Kit provided CT values comparable to those achieved with the RNeasy Mini Kit and QuantiTect Probe RT-PCR Kit.|The FastLane Kit gave lower CT values in real-time one-step RT-PCR than similar kits from other suppliers. Each kit was used with an assay for PPIA (an isomerase).|Real-time one-step RT-PCR analysis of the expression of NFκB1 (nuclear transcription factor) using the FastLane Kit gave reproducible CT values from a 384-well plate of HeLa cells.|The FastLane Kit enabled linear detection of β-actin transcript in 1–10,000 HeLa cells (serial dilutions of lysate were analyzed). In contrast, a similar kit from Supplier SIII showed no linearity.|
Performance
The FastLane Cell Probe Kit delivers sensitive probe-based detection in real-time one-step RT-PCR without the need for RNA purification (see figure "Sensitive probe detection without RNA purification"). Real-time quantification is more sensitive and linear compared with similar kits (see figures "Greater sensitivity than other kits" and "Linear detection down to a single cell"), and is highly reproducible (see figure "Highly reproducible analysis of a 384-well plate"). In addition, genomic DNA does not interfere with quantification.
Principle

The FastLane Cell Probe Kit uses FastLane technology to prepare cell lysates for real-time one-step RT-PCR. 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.

Procedure

With the FastLane Cell Probe Kit, 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.

The kit contains sufficient reagents for cell lysis in two 96-well cell culture plates and subsequent real-time RT-PCR (50 µl PCR volume). The kit 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 Probe RT-PCR Kit (200) needs to be purchased.

Applications

The FastLane Cell Probe Kit is ideal for experiments which require rapid, high-throughput gene expression analysis, such as:

  • Validation of siRNA-mediated gene knockdown
  • Screening of potential drugs
  • Detection of stimulated gene regulation
Feature
Specifications
Applications Probe-based, real-time, one-step RT-PCR
Enzyme activity Primer annealing
Mastermix Yes
Reaction type One-step, cell lysis, RNA stabilization, DNA digestion
Real-time or endpoint Real time
Sample/target type Cultured cells
Single or multiplex Single
With/without hotstart Without hotstart

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Kit Handbooks
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For real-time, one-step RT-PCR analysis of cultured cells without RNA purification
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