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

S_1258_GEF_PCR0393

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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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$836.00
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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" がん研究において最高の感度を実現"、" 再現性の高いcDNA調製")。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

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

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

Specifications

FeaturesSpecifications
ApplicationsReal-time, two-step RT-PCR, gene expression analysis directly from cells
With/without hotstartWithout hotstart
Enzyme activityReverse transcription
Reaction typecDNA production, DNA digestion
MastermixNo
Real-time or endpointReal-time, two-step RT-PCR
Sample/target typeCultured cells
Single or multiplexSingle

Resources

キットハンドブック (1)
For high-speed preparation of first-strand cDNA directly from cultured cells without RNA purification. For use in real-time, two-step RT-PCR
パンフレット (1)
MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
Safety Data Sheets (1)
Certificates of Analysis (1)
Gene Expression Analysis (1)
Kit Handbooks (1)
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

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