荧光定量PCR实验设计及优化.ppt

* 逆转录相关试剂（盒） Transcriptor reverse transcriptase 能够逆转录合成长达14kb的cDNA (full length cDNA) 热稳定性高(工作温度高达65度)，能够逆转录复杂RNA模板 (e.g., GC-rich RNA templates) 具有RNase H 的活性，降解RNA : DNA 杂合链中的RNA * Transcriptor 1st Strand cDNA Synthesis Kit Transcriptor reverse transcriptase 随机引物(6碱基) 和 anchored oligo(dT)18 primers Protector RNase inhibitor 广谱抑制Rnase 适用于pH5.0-9.0; 25oC-55oC 对照 RNA （来源于K562细胞系）和 一对对照 PBGD 引物（胆色素原脱氨酶） anchored oligo (dT)18 primers non-anchored oligo (dT)20 primers N: A/T/G/C; V: A/C/G; B: C/G/T Doing now what patients need next * 我们都知道细胞中含有的RNA含量是非常少的，一般来讲在107个细胞中RNA总共也只有10ug左右，在RNA含量相对丰富的肝组织中要得到10ug的RNA也需要30mg肝组织，应用幼嫩的植物叶片组织，如大豆叶片，100mg左右也能得到10ug左右的RNA。而且在所提取的RNA中我们需要的仅是mRNA，只占RNA总量的1～5％左右，大部分是rRNA，占到80～85％，也就是我们电泳看到的28s和18s，还有一部分tRNA占10％左右。 大家可以看一下右面这张图，象小蘑菇一样的是核糖体，由蛋白和核糖体RNA组成，是蛋白质翻译的场所，象三叶草一样的是转运RNA，由它来负责运输氨基酸，信使RNA则携带有编码氨基酸序列的遗传密码子，可以看到核糖体RNA非常丰富，而带有polyA尾巴的信使RNA只占有很少的比例，同时在细胞中还有丰富的RNA酶。 * What is required for the reverse transcription of RNA into cDNA. Sequence-specific primers selectively prime the mRNA of interest, that means that sequence information is needed. Using this priming method one is able to select for a particular RNA. This type of primer has been used very successfully in diagnostic assays. Sequence specific primers are the only type of priming that can be used for one-step RT-PCR applications. If the fragment of interest is located on the 5‘ end, then a mix of random hexamer primers and anchored oligo(dT)N can give optimal results. Typically 60 μM of random hexamers and 2.5 μM anchored oligo(dT)N are used. The appropriate incubation buffer guarantees an optimal pH and salt concentration for the reverse transcription. Primers are needed to initiate cDNA synthesis. For the elongation of the primer the enzyme needs dNTPs. To catalyze the reaction the reverse transcription enzyme is required. Optional a RNase inhibitor can be added. Especially if the risk of contamination of the template with RNases exists. Depending on the assay,