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FastLane Cell RT-PCR Kits

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
FastLane Cell Probe Kit (200)

Cat. No. / ID: 216413

FastLane Cell One-Step Buffer Set, 2x QuantiTect Probe RT-PCR Master Mix, and QuantiTect RT Mix
detectiontype
Probe
Multiplex Probe
SYBR Green
FastLane Cell RT-PCR Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

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 (see figure " Sensitive detection without RNA purification"). Results in high-throughput experiments are very reproducible (see figure " Highly reproducible analysis of a 96-well plate") 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 (see figure " 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.
With FastLane Cell Multiplex Kits, multiplex analysis in 96- and 384-well formats is highly reproducible (see figure " Highly reproducible multiplex analysis of a 96-well plate"). The kits are ideal for rapid, high-throughput gene expression analysis, such as validation of siRNA-mediated gene knockdown (see figure " Reliable validation of gene silencing"). Target and control genes are amplified in the same reaction with similar, high PCR efficiencies, enabling relative quantification by the ΔΔCt method.

See figures

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 (see figure " Multiplex analysis with no optimization steps") 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.

See figures

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

Comparison of FastLane Cell RT-PCR Kits

Features FastLane Cell SYBR Green Kit FastLane Cell Probe Kit FastLane Cell Multiplex Kits
Applications SYBR Green-based, real-time, one-step RT-PCR Probe-based, real-time, one-step RT-PCR Multiplex, real-time, one-step RT-PCR
Enzyme activity Primer annealing Primer annealing Primer annealing
Master mix Yes Yes Yes
Reaction type One-step, cell lysis, RNA stabilization, DNA digestion One-step, cell lysis, RNA stabilization, DNA digestion One-step, cell lysis, RNA stabilization, DNA digestion
Real time or endpoint Real time Real time Real time
Sample/target type Cultured cells Cultured cells Cultured cells
Single or multiplex Single Single Multiplex
With/without hotstart Without hot start Without hot start Without hot start

Supporting data and figures

Resources

Kit Handbooks (1)
For real-time, one-step RT-PCR analysis of cultured cells without RNA purification
Gene Expression Analysis (1)

FAQ

Is it possible to purify viral RNA using FastLane Cell RT-PCR Kits?
No, the kits are not designed for the purification of viral RNA.
FAQ ID -1484
Which cell lines have been tested with FastLane Cell RT-PCR Kits?

We have successfully tested the following cell lines:

 

Adherent cells Suspension cells Primary cells
HeLa ACC Jurkat HUVEC
HepG2   UASMC*
MCF7   NHBE†

* UASMC: Umbilical artery smooth muscle cells.

† NHBE: Normal human bronchial epithelial cells.

FAQ ID -1485
Can I use more than 4 µl FastLane lysate in subsequent real-time RT-PCR?
Increasing the volume of FastLane lysate will not necessarily increase PCR sensitivity and may, depending on the cell line used, reduce the linearity of the reaction.
FAQ ID -1480
Can I use specific primers with the FastLane Kits?
Yes, specific primers can be used at 0.5 to 1 uM concentration.
FAQ ID -3078
Can the FastLane RT reaction be incubated longer than 30 min at 42 deg C?
FAQ ID -3079
Can the FastLane Cell lysates (containing stabilized RNA, step 8. in Fastlane protocol) be stored?

Yes, the lysate can be stored at -80 deg C.  We have stability data for up to 32 months. It is also fine to freeze and thaw the lysate up to 3 times. The lysate can also be stored at room temperature for up to 2 hours.

FAQ ID -3077
Can I follow the reaction setup for probe-based assays described in Appendix D of the FastLane Cell RT-PCR Handbook using 384-well plates?

In our experience, Q-Solution has no positive effect on probe-based real-time RT-PCR.

 

 

FAQ ID -1482