FastLane Cell cDNA Kit

快速制备用于定量RT-PCR的cDNA，无需RNA纯化

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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)

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FastLane Cell cDNA Kit 旨在用于分子生物学应用。该产品不能用于疾病诊断、预防和治疗。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

由细胞制备cDNA只需四个步骤，45分钟内即可完成
高灵敏度检测低丰度转录产物
易于同时处理多个样本
整合的gDNA去除步骤确保只检测RNA
无需设计RNA特异性引物或探针

Product Details

FastLane Cell cDNA Kit提供由培养细胞直接制备cDNA的方法，操作快速简单，无需纯化RNA。试剂盒提供洗涤和裂解缓冲液，以获得含有RNA的裂解液，同时应用gDNA Wipeput Buffer可去除基因组DNA污染，同时提供用于快速、高效合成cDNA的所有反应组分。该试剂盒适用于需要快速、高通量进行基因表达分析的实验。

Performance

FastLane Cell cDNA Kit制备的cDNA可以使两步法定量PCR得到高灵敏度、高重现性的检测结果（参见""和""）。高品质的cDNA同样可用于对大于10⁵个细胞到单个细胞进行检测。

See figures

Superior sensitivity in cancer research.

Highly reproducible cDNA preparation.

Principle

FastLane Cell cDNA Kit使用FastLane技术可在45分钟内直接由培养细胞制备得到cDNA。为确保real-time RT-PCR只检测到RNA，可使用创新的gDNA Wipeout Buffer去除基因组DNA（参见" Effective elimination of genomic DNA contamination"）。无需设计RNA特异性引物或探针，节约时间和精力。

FastLane Cell cDNA Kit配合优化的QuantiTect、QuantiFast或Rotor-Gene Kits进行real-time RT-PCR，可在数小时内轻松处理并分析细胞样本。由此可对实验的若干转录产物进行快速检测，例如验证siRNA介导的基因沉默效果（参见" Successful analysis of CDC2 knockdown"）。

FastLane Cell cDNA Kit是FastLane Kits的一部分，可加快并实现无缝的基因表达分析流程。

See figures

Effective elimination of genomic DNA contamination.

Successful analysis of CDC2 knockdown.

Procedure

FastLane Cell cDNA Kit直接由培养细胞制备cDNA，只需4个步骤即可在45分钟内获得cDNA：细胞洗涤、细胞裂解并稳定RNA、基因组DNA去除和合成cDNA。制备的cDNA可直接用于两步法real-time RT-PCR。

该试剂盒提供足量用于24孔细胞培养板或48孔细胞培养板制备cDNA的试剂，如果应用96孔细胞培养板，需要额外购买QuantiTect Reverse Transcription Kit（50）。

FastLane Cell cDNA Kit制备的cDNA，可使用下列QuantiTect Kits中的一种通过两步法real-time RT-PCR进行分析：

SYBR^® Green检测：QuantiTect SYBR^® Green PCR Kit
探针：QuantiTect Probe PCR Kit
多重探针检测：QuantiTect Multiplex PCR Kits

FastLane Cell cDNA Kit配合QuantiFast Kit使用可进行快速的两步法real-time RT-PCR：

SYBR^® Green检测：QuantiFast SYBR^® Green PCR Kit
探针检测：QuantiFast Probe PCR Kits
多重探针检测：QuantiFast Multiplex PCR Kits

使用Rotor-Gene Q实时荧光定量PCR分析仪对FastLane Cell cDNA Kit制备的cDNA进行分析时，应用如下Rotor-Gene Kits可进行超快速的两步法real-time RT-PCR：

SYBR^® Green检测：Rotor-Gene SYBR^® Green PCR Kit
探针检测：Rotor-Gene Probe PCR Kit
多重探针检测：Rotor-Gene Multiplex PCR Kits

QuantiTect及QuantiFast Kits与多种real-time热循环仪兼容，包括Applied Biosystems提供的仪器。

Applications

FastLane Cell cDNA Kit适用于需要对在各转录本水平进行快速检测的实验，包括:

验证siRNA介导的基因沉默效果
评估药物效果
基因调控检测

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

Download Safety Data Sheets for QIAGEN product components.

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