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FastLane Cell cDNA Kit

For high-speed preparation of cDNA without RNA purification for use in real-time RT-PCR

Features

  • 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

Useful Resources

FastLane Cell cDNA Handbook - (EN) (EN) - FastLane Kits — from Sample Direct to Result
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Superior sensitivity in cancer research.
Successful analysis of CDC2 knockdown.
Highly reproducible cDNA preparation.
Effective elimination of genomic DNA contamination.
FastLane Cell cDNA Kit (50)

Cat. No. / ID: 215011

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)
 $675.00
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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.

Product Details

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.

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.
See figures
Superior sensitivity in cancer research.Superior sensitivity in cancer research.Highly reproducible cDNA preparation.Highly reproducible cDNA preparation.

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.

See figures
Effective elimination of genomic DNA contamination.Effective elimination of genomic DNA contamination.Successful analysis of CDC2 knockdown.Successful analysis of CDC2 knockdown.

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

Supporting data and figures

Superior sensitivity in cancer research.

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.
Superior sensitivity in cancer research.
Superior sensitivity in cancer research.
Successful analysis of CDC2 knockdown.
Highly reproducible cDNA preparation.
Effective elimination of genomic DNA contamination.

Specifications

FeaturesSpecifications
applicationsReal-time, two-step RT-PCR, gene expression analysis directly from cells
withwithouthotstartWithout hotstart
enzymeactivityReverse transcription
reactiontypecDNA production, DNA digestion
mastermixNo
realtimeorendpointReal-time, two-step RT-PCR
sampletargettypeCultured cells
singleormultiplexSingle

Resources

Kit Handbooks (1)
FastLane Cell cDNA Handbook - (EN)
PDF (198KB)
For high-speed preparation of first-strand cDNA directly from cultured cells without RNA purification. For use in real-time, two-step RT-PCR
Gene Expression Analysis (1)
(EN) - FastLane Kits — from Sample Direct to Result
PDF (432KB)

FAQ

Is there a stopping point in the protocol for Suspended Cells using the FastLane Cell cDNA Kit?

Yes. Cells can be frozen after the wash step with Buffer FCW in Appendix D Protocol 'Processing Suspended Cells' of the FastLane Cell cDNA Handbook. Buffer FCW should be aspirated promptly after pelleting the cells during the wash step. Following the removal of Buffer FCW at step D5, the cells can be frozen. For future processing, thaw the cell pellet, and continue with the lysis step D6 using Buffer FCP.

FAQ ID -837
Can the FastLane Cell cDNA Kit also be used for qualitative end-point PCR?
Unfortunately, the FastLane Cell cDNA Kit cannot be used for classic end-point PCR. It is optimized for quantitative two-step RT-PCR, and can only be used for this application.
FAQ ID -834
Is it possible to use frozen cell pellets with the FastLane Cell cDNA Kit?

Yes, it is possible to process frozen cell pellets with the FastLane Cell cDNA Kit. Follow Appendix D protocol 'Processing Suspended Cells' in the FastLane Cell cDNA Handbook, starting from step D3. In our R&D labs, we let the pellet stand for 30 seconds at room temperature, add wash buffer FCW, spin down the cells promptly, and aspirate the wash buffer. In general, cells should not be vortexed or pipetted up and down during the wash step, and they should not be incubated in buffer FCW.

Note that it is essential to wash the cells with Buffer FCW at step D3. Omitting this step, or washing with PBS only, will inhibit subsequent steps in the protocol.

 

FAQ ID -835
Can I use my gene-specific primers with the FastLane Cell cDNA Kit and the QuantiTect Reverse Transcription Kit?

Yes, you can substitute the RT Primer Mix supplied in the FastLane Cell cDNA Kit and the QuantiTect Reverse Transcription Kit with your gene-specific primers. We suggest optimizinig the primer concentration by titration, starting at 1 uM, and gradually decreasing it to 0.5 uM final concentration in the reaction. Optimal amounts will depend on the specific primers you are using.

FAQ ID -812
How do the FastLane Cell cDNA Kit and QuantiTect Reverse Transcription Kit eliminate genomic DNA contamination?
The FastLane Cell cDNA Kit and QuantiTect Reverse Transcription Kit contain a unique buffer, called gDNA Wipeout Buffer, which ensures complete removal of gDNA after a brief incubation step.
FAQ ID -783
How many cells can be used with the FastLane Cell cDNA Kit?

A range of 1 x 104 - 1 x 105 cells can be used with the FastLane Cell cDNA Kit, depending on the culture plate format. Different plate formats require different amounts of buffers FCW and FCP. Refer to Appendix C, Table 4 in the FastLane Cell cDNA Handbook for the number of cells to seed per well and the buffer volumes to use. Note that the table is a starting point for optimization providing suggestions for cell numbers and buffer volumes. The number of cells to seed per well depends on factors such as cell type and culture conditions. For optimal results in real-time RT-PCR, it may be necessary to optimize the number of cells and buffer volumes.

FAQ ID -796
What cell lines have been tested with the Fastlane Cell cDNA Kit?

We have used the Fastlane Cell cDNA Kit successfully with the following cell lines in-house:

Human

  • HEK293
  • K562
  • HepG2
  • Hela ACC
  • Hela S3
  • MCF7
  • Jurkat
  • HUVEC

Mouse

  • NIH3T3

In addition, we have customer information that the kit works fine with:

  • Burkitt Lymphoma cell line (BL2)
  • Neuroblastoma cell line
  • Prostate carcinoma cell line

According to customer reports, the FastLane Cell cDNA Kit does not work with Osteoblasts.

FAQ ID -1175
Will the FastLane Cell cDNA Kit work with suspension cells?
Yes, the FastLane Cell cDNA Kit can be used for suspension cells. A complete protocol can be found in Appendix D of the FastLane Cell cDNA Handbook.
FAQ ID -801
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
What is the cell number range one can use with the FastLane Cell cDNA Kit for real-time RT-PCR?

The FastLane Cell cDNA Kit enables accurate detection over a wide linear range from 1 x 105 cells down to one cell in real-time RT-PCR. Data is shown on our website in the figure 'Linear Detection in Real-Time RT-PCR Down to One Cell'.

For optimized FastLane Buffer volumes (FCW and FCP) per cell number seeded, please see Appendix C of the FastLane Cell cDNA Handbook: 'Seeding and Processing Cells for Different Plate Formats'.

FAQ ID -782
Can the FastLane Cell cDNA Kit be used with tissue?
No, the FastLane Cell cDNA Kit cannot be used with tissue. The lysis conditions are insufficient for tissue samples.
FAQ ID -821

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