Q1：无Ct值出现？

A1：

1) 采集荧光信号的步骤有误。检查程序设置，两步法扩增在退火延伸步骤采集信号，三步法扩增在延伸阶段采集信号；

2) 引物或模板降解。PAGE电泳检测引物完整性，琼脂糖凝胶电泳检测模板DNA或RNA的质量及完整性，RNA模板建议重新逆转录获得cDNA，cDNA应尽快使用；

3) 模板量不足。适当增加模板用量或重新制备高浓度模板。

Q2：实验重复性差？

A2：

1) 加样存在误差。使用性能较好的移液枪，配制预混液后分装，减少移液误差；

2) 荧光定量PCR仪器不同位置温度控制不一致。定期校准仪器；

3) 模板浓度低或拷贝数低。适当增加模板用量。

Q3：扩增曲线形状异常？

A3：

1) 扩增曲线不光滑。信号太弱，经系统矫正后产生，可提高模板浓度重复实验；

2) 扩增曲线断裂、骤降。模板浓度较高或反应管内留有气泡,减小基线终点(CT值-4)重新分析数据，或上机前离心并仔细检查反应管内是否有气泡残留；

Q4：同一试剂在不同仪器上产生不同的曲线？

A4：

分析扩增效率，灵敏度，特异性，如果扩增效率在 90%~110%，且都是特异性扩增，则数据均可分析使用。

说明书：点击下载

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