实时荧光定量PCR实验结果分析讲解.ppt

* AquaPure RNA 分离试剂盒: 无需接触有毒的苯、胍或氯仿等试剂 高速度— 从细胞、组织或血液到纯化的RNA 只需大约30 min 应用广泛的操作流程— 液相操作流程适合范围极广的应用领域 * 同一个管中同时扩增目标基因和看家基因，看家基因作为内标对目标基因进行校正，以消除反转录过程以及样品制备过程中产生的人为差异对定量 结果的影响。 * 假设我们知道样本管中含有500,000个p53 mRNA分子，但是并不清楚其生物学上的意义，生物学家们更加关注如下的陈述：1）一定量血样中病毒颗粒的数目，或2）比较正常组织，等量的癌组织中p53 mRNA的倍数发生改变。 * Relative quantification. Refers to quantifying samples against one another to get final fold, or relative, amounts. Just as in an absolute quantification assay, you should have a standard reference curve. This curve will serve to establish the efficiency of the assay thereby allowing a more accurate comparison between samples. The simplest way to generate a relative standard curve is to simply generate a dilution series. You don’t need a 10 or 100 fold dilution series, a simple 3 or 4 fold one will do just fine. You can actually make the dilution series from one of the samples, plasmid of pre-amplified DNA (in reality the source is not critically important). You do want to make sure that the standards mimics the samples, so if you know your sample is in Buffer X, it’s a good idea to make your dilution series in Buffer X. If you are quantifying genomic DNA, use genomic DNA as your standard or pike some non specific DNA into plasmid or other targets. The idea is to prepare standard and samples such that we end up comparing “oranges” to “oranges”. Also important is the no template control… * At this point, we set the threshold and look at three unknowns, and collect their CT values. It’s important to remind customers that both standards and unknowns will be amplifying at the same time. * These CT’s are plotted against the standard curve giving us values relative to the standard curve. These values (the scale is arbitrary) can then be compared to one another to give us a “fold” comparison. * 如 * It’s important to note that some customers run a reference (such as Actin) standard curve and then compare the unknowns to that standard curve. This is not correct! Samples f