FastLane Cell cDNA Kit

RNA精製なしにリアルタイムRT-PCRに使用できるcDNAを迅速に調製

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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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￥85,500

FastLane Cell cDNA Kitは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

わずか4ステップ、45分以下で細胞からcDNAを精製
発現量の低い転写物でも高感度で検出
複数のサンプルを簡単に同時処理可能
gDNA除去が組み込まれているため、RNAだけを検出可能
RNA特異的なプライマーやプローブのデザイン不要

Product Details

FastLane Cell cDNA Kitは、RNA精製を行なわずに直接培養細胞から迅速で簡単な一本鎖cDNAの調製を実現します。本キットにはライセート調製とRNA安定化のための洗浄および溶解バッファー、混入したゲノムDNA除去用gDNA Wipeout Buffer、迅速で効率的なcDNA合成用反応液が全て含まれています。FastLane Cell cDNA Kitは迅速でハイスループットな遺伝子発現解析が必要な実験に最適です。

Performance

FastLane Cell cDNA Kitで合成したcDNAは2ステップリアルタイムRT-PCRで感度および再現性の高い結果を実現します（図""、""）。この高品質cDNAを用いて幅広い細胞数（1から10⁵以上）の範囲でも検出を行なうことができます。

See figures

Superior sensitivity in cancer research.

Highly reproducible cDNA preparation.

Principle

FastLane Cell cDNA KitはFastLane技術を利用し、培養細胞からわずか45分で直接cDNAを調製します。リアルタイムRT-PCRにおいてRNAのみを確実に検出するため、ゲノムDNAの除去に革新的なgDNA Wipeout Bufferを使用しています（図" ゲノムDNAコンタミを効率的に除去"）。RNA特異的なプライマーあるいはプローブをデザインする必要がなく、時間および労力を節約できます。

リアルタイムRT-PCRに至適化済みのQuantiTect、QuantiFast、またはRotor-Gene KitsをFastLane Cell cDNA Kitと共に使用することにより、複数の細胞サンプルを数時間以内に容易に処理し、解析することができます。従って、siRNAによる遺伝子発現ノックダウンの検証のような実験において、複数の転写物のスナップショットが可能です（図" CDC2ノックダウンの優れた解析結果"）。

FastLane Cell cDNA Kitは、遺伝子発現解析ワークフローのスピードアップと能率化を実現するFastLane製品の1つです。

See figures

Effective elimination of genomic DNA contamination.

Successful analysis of CDC2 knockdown.

Procedure

FastLane Cell cDNA Kitを用いて、細胞洗浄、RNAの安定化を組み込んだ細胞溶解、ゲノムDNAの除去、cDNA合成の4ステップの操作で、培養細胞からわずか45分でcDNAが得られます。合成されたcDNAは2ステップリアルタイムRT-PCRに即使用できます。

本キットには24ウェル細胞培養プレート2枚、あるいは48ウェル細胞培養プレート1枚でcDNAを調製するために十分な量の試薬が入っています。96ウェル細胞培養プレートで調製を行なう場合には、QuantiTect Reverse Transcription Kit（50）を追加購入する必要があります。

FastLane Cell cDNA Kitで調製したcDNAは、以下のQuantiTect Kitシリーズの1つを用いて2ステップリアルタイムRT-PCRで解析できます。

SYBR^® Green検出：QuantiTect SYBR^® Green PCR Kit
プローブ検出：QuantiTect Probe PCR Kit
マルチプレックスプローブ検出：QuantiTect Multiplex PCR Kits

あるいは、FastLane Cell cDNA KitをQuantiFast Kitと組み合わせて2ステップ高速リアルタイムRT-PCRを行なうことも可能です。

SYBR^® Green検出：QuantiFast SYBR^® Green PCR Kit
プローブ検出：QuantiFast Probe PCR Kits
マルチプレックスプローブ検出：QuantiFast Multiplex PCR Kits

FastLane Cell cDNA Kitで調製したcDNAを、以下のような専用Rotor-Gene Kitを用いてRotor-Gene Qサイクラーで解析すると、超高速2ステップリアルタイムRT-PCRを行なうことができます。

SYBR^® Green検出：Rotor-Gene SYBR^® Green PCR Kit
プローブ検出：Rotor-Gene Probe PCR Kit
マルチプレックスプローブ検出：Rotor-Gene Multiplex PCR Kit

QuantiTectおよびQuantiFast KitはApplied Biosystems社の装置を含む様々なリアルタイムサイクラーで使用可能です。

Applications

FastLane Cell cDNA Kit は以下のような実験に適しています。

siRNAによる遺伝子ノックダウンの検証
薬剤効果の評価
遺伝子制御の検出

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