FastLane Cell cDNA Kit

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

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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)

836,00 US$

Quantity

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.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

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

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 "" and ""). 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.

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.

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 C_T values were achieved when cDNA was prepared with the FastLane Kit instead of a similar kit from Supplier A_IV.

Specifications

Features	Specifications
Applications	Real-time, two-step RT-PCR, gene expression analysis directly from cells
With/without hotstart	Without hotstart
Enzyme activity	Reverse transcription
Reaction type	cDNA production, DNA digestion
Mastermix	No
Real-time or endpoint	Real-time, two-step RT-PCR
Sample/target type	Cultured cells
Single or multiplex	Single

Resources

For high-speed preparation of first-strand cDNA directly from cultured cells without RNA purification. For use in real-time, two-step RT-PCR

FAQ

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

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

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

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

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

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

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

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

Yes, specific primers can be used at 0.5 to 1 uM concentration.

FAQ ID -3078

FAQ ID -3079

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

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

No, the FastLane Cell cDNA Kit cannot be used with tissue. The lysis conditions are insufficient for tissue samples.

FAQ ID -821