When synthesizing cDNA for real-time PCR, the PrimeScript RT Reagent Kit (Perfect Real Time) allows maximal flexibility during reaction assembly. This reverse transcription reagent kit facilitates cDNA synthesis prior to analysis by real-time PCR. Designed for two-step real-time RT-PCR (also called qRT-PCR or RT-qPCR), the PrimeScript RT Reagent Kit (Perfect Real Time) contains all of the components needed for reverse transcription, including PrimeScript RTase, RNase inhibitor, random 6-mers, oligo dT primer, dNTPs, and reaction buffer. Because the kit"s primers and RTase are provided as separate components, there is greater flexibility in reaction assembly.
When synthesizing cDNA for real-time PCR, the PrimeScript RT Reagent Kit (Perfect Real Time) allows maximal flexibility during reaction assembly. This reverse transcription reagent kit facilitates cDNA synthesis prior to analysis by real-time PCR. Designed for two-step real-time RT-PCR (also called qRT-PCR or RT-qPCR), the PrimeScript RT Reagent Kit (Perfect Real Time) contains all of the components needed for reverse transcription, including PrimeScript RTase, RNase inhibitor, random 6-mers, oligo dT primer, dNTPs, and reaction buffer. Because the kit"s primers and RTase are provided as separate components, there is greater flexibility in reaction assembly.
The PrimeScript RT Reagent Kit (Perfect Real Time) includes PrimeScript reverse transcriptase for strong strand-displacement activity and efficient synthesis of cDNA for real-time PCR. PrimeScript RTase is a modified, recombinant MMLV (Moloney murine leukemia virus) reverse transcriptase and is verified to be RNase H-minus. PrimeScript RTase is also robust, versatile, and can be used for reverse transcription of virtually any RNA template, including templates that have a high degree of secondary structure or are GC rich. Because of the excellent extension capability of PrimeScript, cDNA synthesis reactions can be performed at 42°C, minimizing the risk of RNA degradation that often occurs at higher temperatures.
If a master mix is preferred, consider using PrimeScript RT Master Mix (Perfect Real Time). Use this kit in combination with one of Takara Bio"s qPCR premix products, such as TB Green Premix Ex Taq II (Tli RNase H Plus) (Cat. # RR820A), TB Green Premix Ex Taq (Tli RNase H Plus) (Cat. # RR420A), or Premix Ex Taq (Probe qPCR) (Cat. # RR390A), for intercalating green dye detection or 5"-nuclease probe-mediated assays.
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TAKARA的RR047A逆转录试剂盒，做逆转录程序设置错误本来应该37℃15min85℃5s，变成37℃15s，85℃
5s，请问再重新设置程序可以吗，85℃酶会全部失活吗？
加尾法是采用加A酶先对mirna进行加尾，然后再用带oligodt的引物反转录，他的反转录引物是通用的，一次反转录可以获得所有miRNA的cdna，效率高。茎环法是采用特异性反转录引物序列+颈环结构作为反转录引物进行反转录的，一次反转录只能获得一种mirna的cdna，可能有几种一起反转录的，这个我不太清楚一起反转录的效果。然后茎环法的经典即ABI的探针法定量试剂盒。我用过茎环法反转录+染料法定量的，下游引物为通用引物，结果不太好，就现在用的这家的他采用的是双向引物都是特异性的，效果还可以。不知道我说的清楚不，希望能对你有帮助。
向左转|向右转
逆转录（reverse transcription）是以RNA为模板合成DNA的过程，即RNA指导下的DNA合成。此过程中，核酸合成与转录（DNA到RNA）过程与遗传信息的流动方向（RNA到DNA）相反，故称为逆转录。逆转录过程是RNA病毒的复制形式之一，需逆转录酶的催化。 逆转录过程的揭示是分子生物学研究中的重大发现，是对中心法则的重要修正和补充。人们通过体外模拟该过程，以样本中提取的mRNA为模板，在逆转录酶的作用下，合成出互补的cDNA，构建cDNA文库，并从中筛选特异的目的基因。该方法已成为基因工程技术中最常用的获得目的基因的策略之一。
大家miRNA逆转录和qPCR的试剂盒用的是那个公司的啊，求推荐！
