反转录PCR (reverse transcription PCR, RT-PCR),是指以mRNA为模板在反转录酶作用下合成的cDNA 第一链的PCR。该反应分为两步,第一步在单引物的介导和反转录酶的催化下合成RNA 的互补链cDNA; 第二步加热后cDNA 与RNA 链解离,然后与另一引物退火,并由DNA 聚合酶催化引物延伸生成双链DNA, 最后同常规PCR一样,...
Protocol for MasterAmp™ High Fidelity RT-PCR KitHigh, MasterampKit, Fidelity Rtpcr
For quantifying mRNA, we use a competitiveRT-PCRprotocol with internal standard RNAs. These are added in a defined quantity to the RNA sample prior to the RT reaction. The resulting standard cDNAis coamplified with the same primers as the endogenous target sequence. Its PCR product is approxim...
This RT- PCR technique was performed on sectioned tissues of female buds of the cucumber GY3 inbred line. The gene, encoding the MADS-box type (agamus-like) protein, the expression pattern of which was described earlier [10], was used as a marker gene for optimisation of steps in the ...
real-time RT-PCR中间的"RT"是 reverse transcripti on(逆转录),整个意思就是利用mRNA或总R NA为模板的实时逆转录PCR技术(quantitative R eal-time RT-PCR)。real time RT-PCR指的是qPC R+RT-PCR的组合,是说将mRNA逆转录为cDNA后再作为模板进行实时荧光PCR分析,因为RT-PCR是可以定性的,但不能进行定量检测...
We will provide both one-step and two-step protocols for RT-PCR. We recommend the two-step protocol for this class. In the one-step protocol, the components of RT and PCR are mixed in a single tube at the same time. The one-step protocol generally works well for amplifying targets tha...
Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 渭l of ...
RT-PCR protocolRT-PCR步骤 RNA提取(-70℃保存) 1.六孔板每孔细胞加入1ml Trizol,冰上放置5min,枪头吹打 2.将裂解液1ml转入一新EP管中(1.5ml),加氯仿0.2ml,剧烈振荡混合,室温放置5min,10000 rpm 4℃离心15min 3.转移上层水相(吸70%)到一新EP管中,加入等体积的异丙醇(约0.5ml),振荡混合,室温保存10...
RT-PCRprotocol RT-PCR 实验步骤 一、总RNA提取 1. 取200mg组织，放到1.5mlEP管中，加入1mlTrizol剪碎。 2. 震荡30s。 3. 加0.2ml氯仿，剧烈摇动30s，室温3min。 4. 12000×g，4℃离心，15min。 5. 吸上层无色水相，移入另一EP管中（约0.5ml）。 6. 加等体积异丙醇...
【简要说明】:此文档讲述了普通RT-PCR的操作步骤(Protocol)。 【下载地址】: http://pan.baidu.com/s/1dDF0YY1