For high-speed preparation of cDNA without RNA purification for use in real-time RT-PCR
  • From cells to cDNA in only 4 steps and under 45 minutes
  • Sensitive detection of even low-abundance transcripts
  • Easy parallel processing of several samples
  • Integrated gDNA removal allows detection of RNA only
  • No need to design RNA-specific primers or probes

The FastLane Cell cDNA Kit provides a fast and simple procedure for preparing first-strand cDNA directly from cultured cells without RNA purification. The kit is supplied with wash and lysis buffers for preparing lysates and stabilizing RNA, gDNA Wipeout Buffer for eliminating genomic DNA contamination, and all reaction components for fast and efficient cDNA synthesis. The FastLane Cell cDNA Kit is ideal for experiments requiring rapid, high-throughput gene expression analysis.

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Quantity Product Cat. no. Price Sum
 
FastLane Cell cDNA Kit (50)
Buffer FCW, Buffer FCP, and components for 50 x 20 µl reverse-transcription reactions (gDNA Wipeout Buffer, Quantiscript Reverse Transcriptase, Quantiscript RT Buffer, RT Primer Mix, and RNase-Free Water)
215011
$583.00
$0.00
The FastLane Cell cDNA 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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Superior sensitivity in cancer research.|Successful analysis of CDC2 knockdown.|Effective elimination of genomic DNA contamination.|Highly reproducible cDNA preparation.|
In real-time two-step RT-PCR analysis of various genes important to cancer research in HeLa cells, lower CT values were achieved when cDNA was prepared with the FastLane Kit instead of a similar kit from Supplier AIV.| MCF-7 cells were transfected with various CDC2 siRNAs (S1, S2, or S3) or nonsilencing siRNA (NS). After 48 hours, cDNA was prepared using the FastLane Kit. Expression of CDC2 (cell cycle protein) relative to GAPDH (endogenous control) was analyzed by real-time two-step RT-PCR. The relative expression of CDC2 in untreated cells (UT) was set to 100%. |cDNA was prepared using the FastLane Cell cDNA Kit, with (+RT) or without (-RT) reverse transcriptase. The cDNA was quantified using a gene expression assay for β-actin. The -RT plot in indicates that no genomic DNA was detected.|cDNA was prepared from a 48-well plate containing HeLa cells using the FastLane Kit. Expression of tubulin B (structural protein) was analyzed by real-time two-step RT-PCR.|
Performance
cDNA synthesized by the FastLane Cell cDNA Kit enables highly sensitive and reproducible results in real-time two-step RT-PCR (see figures "Superior sensitivity in cancer research" and "Highly reproducible cDNA preparation"). The high quality of the cDNA also allows detection over a wide linear range from >105 cells down to one cell.
Principle

The FastLane Cell cDNA Kit uses FastLane technology to prepare cDNA directly from cultured cells in just 45 minutes. To ensure only RNA is detected in real-time RT-PCR, novel gDNA Wipeout Buffer is used to eliminate genomic DNA (see figure "Effective elimination of genomic DNA contamination"). Time and effort are saved as there is no need to design RNA-specific primers or probes.

When the FastLane Cell cDNA Kit is used together with optimized QuantiTect, QuantiFast, or Rotor-Gene Kits for real-time RT-PCR, several cell samples can be easily processed and analyzed within a few hours. This allows a snapshot of several transcripts in experiments such as validation of siRNA-mediated gene knockdown (see figure "Successful analysis of CDC2 knockdown").

The FastLane Cell cDNA Kit is part of the comprehensive range of FastLane Kits, which speed up and streamline the gene expression analysis workflow.

Procedure

With the FastLane Cell cDNA Kit, cDNA is obtained from cultured cells in just 45 minutes in a 4-step procedure: cell wash; cell lysis with integrated RNA stabilization; genomic DNA elimination; and cDNA synthesis. The synthesized cDNA is ready to use in real-time two-step RT-PCR.

The kit contains sufficient reagents for preparing cDNA from two 24-well cell-culture plates or one 48-well cell-culture plate. If working with a 96-well cell-culture plate, an additional QuantiTect Reverse Transcription Kit (50) needs to be purchased.

cDNA prepared with the FastLane Cell cDNA Kit can be analyzed by real-time two-step RT-PCR using one of the following QuantiTect Kits:

  • SYBR® Green detection: QuantiTect SYBR® Green PCR Kit
  • Probe detection: QuantiTect Probe PCR Kit
  • Multiplex probe detection: QuantiTect Multiplex PCR Kits

Alternatively, fast real-time two-step RT-PCR can be carried out when the FastLane Cell cDNA Kit is used in combination with a QuantiFast Kit:

  • SYBR® Green detection: QuantiFast SYBR® Green PCR Kit
  • Probe detection: QuantiFast Probe PCR Kits
  • Multiplex probe detection: QuantiFast Multiplex PCR Kits

Ultrafast real-time two-step RT-PCR can be achieved when cDNA prepared with the FastLane Cell cDNA Kit is analyzed on the Rotor-Gene Q cycler using one of the following dedicated Rotor-Gene Kits:

  • SYBR® Green detection: Rotor-Gene SYBR® Green PCR Kit
  • Probe detection: Rotor-Gene Probe PCR Kit
  • Multiplex probe detection: Rotor-Gene Multiplex PCR Kit 

QuantiTect and QuantiFast Kits are compatible with various real-time cyclers, including instruments from Applied Biosystems.

Applications

The FastLane Cell cDNA Kit is suitable for experiments which require a snapshot of individual transcript levels, such as:

  • Validation of siRNA-mediated gene knockdown
  • Evaluation of drug effects
  • Detection of gene regulation
Feature
Specifications
Applications Real-time, two-step RT-PCR, gene expression analysis directly from cells
Enzyme activity Reverse transcription
Mastermix No
Reaction type cDNA production, DNA digestion
Real-time or endpoint Real-time, two-step RT-PCR
Sample/target type Cultured cells
Single or multiplex Single
With/without hotstart Without hotstart

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Kit Handbooks
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RNA精製を行なわずに直接培養細胞から一本鎖cDNAを迅速に調製
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For high-speed preparation of first-strand cDNA directly from cultured cells without RNA purification. For use in real-time, two-step RT-PCR
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