Q：建议qPCR 实验用几步法？

A：常用 2 步法。需提高扩增特异性，可选用 2 步法或提高退火温度。在扩增效率低， ct 值过大的时候，可以改用 3 步法或延长延伸时间。

Q：预变性 5 min 调整成了 10min，对于实验结果有影响吗？

A：有影响，预变性时间较长可能会影响酶的活性，导致 PCR 产物的产量有所降低。Q：qPCR 实验结果的有效性？为什么建议Ct 值要大于 15？

A：有效性要满足三个条件：（1）标准曲线：扩增效率范围:90-110%，对应斜率为  -3--3.5。 R2＞0.98。 (扩增效率=10-1/斜率-1)，当斜率=-3.32 时，扩增效率=100%。（2）扩 增曲线：S 型曲线，且 Ct 值在 15－35 之间，阴性对照 Ct＞35 或无 Ct 值。（3）熔解曲线：为单一峰。

Ct 值大于 15 个循环是因为 3-15 个循环的荧光值标准差的 10 倍是荧光阈值，Ct 值太小了会影响曲线。

Q：同一基因复孔间熔解曲线 Tm 值有差异？

A：同样的扩增产物也会出现Tm 值有微小差异，一般差异在 1 度以内都可以接受。

Q：为什么稀释了模板CT 值反而变小了？

A:一般 CT 值与模板起始浓度呈负相关，浓度越高，CT 值越小。但也有很多特殊情况， 比如体系中存在抑制物或是模板不纯，这时候稀释模板反而能使 CT 值变低。

Q：内参CT 值小于 20，目的基因均大于 30，怎么办？

A：可能是目的基因为低丰度表达基因导致。建议：a）换用内参；b）换引物；c）换检测线性范围更广的qPCR mix。

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