荧光定量PCR实验数据解决方案.ppt

同时，这些曲线都没有明显的指数增长期。 如何判断它们是否存在扩增？ 其次，看Y轴的数值。 这些“可疑”曲线的指数增长期范围常常是落在1e-2 的范围内。 对数图 如何判断它们是否存在扩增？ 最后，查阅线性图： 曲线一直没有指数上升的趋势，提示不存在扩增。 轻微的荧光增长可能来源于探针的降解。 如何判断它们是否存在扩增？ 案例3:是否存在扩增？ 右侧的曲线形状和扩增曲线很相似，但是曲线不光滑。 切换到线性图，可以看到曲线有一定的上升。 如何判断它们是否存在扩增？ 分析 这些样品在反应的最后阶段才开始扩增，平台期很接近背景。 这些样品的扩增曲线出现了指数扩增的区段 虽然我们可以认为这些样品是临界阳性，但是对这些样品的定量结果都是不太准确的。 形成这类曲线的原因可能是模板的质量差 (RT / PCR抑制物; 模板浓度低)， 也可能是引物探针的设计问题。 试剂原因 如果使用的试剂背景信号太强，荧光的增长将会很小，导致扩增曲线的指数增长期会变得很短，或者没有。 荧光信号差的试剂也会有同样的问题。 总结：遵循扩增曲线形态第一，Ct值第二的判读原则。 查看MultiComponent Plot：是否有正常的阳性扩增曲线 查看扩增曲线的形状：是否有明显扩增阶段（指数增长期）? 指数图下扩增曲线间是否平行? 查看Y轴的数值：扩增曲线最高点的纵坐标值是否符合预期？ 在同一坐标系下查看阳性对照与可疑样品的扩增曲线：避免误判阳性。 咨询联络方式 全国免费技术支持电话： 800 820 8982 或 400 820 8982 技术支持邮箱：CNAPMsupport@ * * How about in this case? A tougher call. Let’s see why. If these are all amplified using the same assay, it’s pretty safe to say that the curves on the right are not real amplification. Unless these samples were extracted in a totally different manner, such that the ones on the right contained MUCH more inhibitor than those on the right, then this is not real amplification. If these are all amplified using the same assay, it’s pretty safe to say that the curves on the right are not real amplification. Unless these samples were extracted in a totally different manner, such that the ones on the right contained MUCH more inhibitor than those on the right, then this is not real amplification. The suspect curves (if indeed they are amplification curves at all) are very close to the noise. As such, they are highly suspect. Also, remind everyone that the Y axis is a LOG scale. So every step up is actually an order of magnitude. This makes the noise look a lot closer to real amplification than it really is. To get a better perspective on things, you can switch to a LINEAR view. There is still a very shallow rise in the suspect samples, but it doesn’t appear to be amplification, as there is no geometric (curving) rise to the suspect samples. We may be seeing some gradual breakdown of the probe