快速制备用于定量RT-PCR的cDNA,无需RNA纯化

  • 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

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。

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FastLane Cell cDNA Kit (50)
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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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.
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Superior sensitivity in cancer research.|Successful analysis of CDC2 knockdown.|Effective elimination of genomic DNA contamination.|Highly reproducible cDNA preparation.|
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.| MCF-7 cells were transfected with various CDC2 siRNAs (S1, S2, or S3) or nonsilencing siRNA (NS). After 48 hours, cDNA was prepared using the FastLane Kit. Expression of CDC2 (cell cycle protein) relative to GAPDH (endogenous control) was analyzed by real-time two-step RT-PCR. The relative expression of CDC2 in untreated cells (UT) was set to 100%. |cDNA was prepared using the FastLane Cell cDNA Kit, with (+RT) or without (-RT) reverse transcriptase. The cDNA was quantified using a gene expression assay for β-actin. The -RT plot in indicates that no genomic DNA was detected.|cDNA was prepared from a 48-well plate containing HeLa cells using the FastLane Kit. Expression of tubulin B (structural protein) was analyzed by real-time two-step RT-PCR.|
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"Superior sensitivity in cancer research"和"Highly reproducible cDNA preparation")。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。
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。 

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

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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
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