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如何稳定保存RNA和定量？

发布: 2017-08-26 2,933阅读 914评论

DNA（RNA）定量分析可用紫外光谱分析，原理是DNA（RNA）分子在260nm处有特异的紫外吸收峰，且吸收强度与DNA(RNA)的浓度成正比。此外，还可通过琼脂糖凝胶电泳上显示的DNA（RNA）带的亮度来分析，因为EB作为一种荧光染料，能插入DNA(RNA)的碱基对平面之间而结合于其上，在紫外光的激发下产生荧光，DNA(RNA)分子上EB的量与DNA分子的长度和数量成正比。在电泳时加入已知浓度的DNA（RNA）Marker作为DNA（RNA）分子量及浓度的参考，样品DNA(RNA)的荧光强度就可以大致 表示DNA（RNA）量的多少。这种方法的优点是简便易行，可结合琼脂糖凝胶电泳分析DNA(RNA)样品的完整性来进行，缺点是不太准确。

1）检测RNA溶液的吸光度
280、320、230、260nm下的吸光度分别代表了核酸、背景（溶液浑浊度）、盐浓度和蛋白等有机物的值。一般的，我们只看OD260/OD280（Ratio，R）。
如果RNA的量够，可在260nm（A260）用分光光度法测定RNA的得率，1个单位等于40ug/mlssRNA。纯RNA的A260/A280的比值为2.0。A260/A230的比值还表明RNA的纯度，其值小于2.0表明裂解液中有亚硫氰胍和belta-巰基乙醇残留，其值大于2.4，需用乙酸盐，乙醇沉淀RNA。

2）RNA的电泳图谱
一般的，RNA的电泳都是用变性胶进行的，但是根据我的经验，如果你仅仅是为了检测RNA的质量是没有必要进行如此麻烦的实验的，用普通的琼脂糖胶就可以了。

电泳的目的是在于检测28S和18S条带的完整性和他们的比值，或者是mRNA smear的完整性。一般的，如果28S和18S条带明亮、清晰、条带锐利（指条带的边缘清晰），并且28S的亮度在18S条带的两倍以上，我们认为RNA的质量是好的。

以上是我们常用的两种方法，但是这两种方法都无法明确的告诉我们RNA溶液中有没有残留的RNA酶。如果溶液中有非常微量的RNA酶，用以上方法我们很难察觉，但是大部分后续的酶学反应都是在37度以上并且是长时间进行的。这样，如果RNA溶液中有非常微量的RNA酶，那么在后续的实验中就会有非常适合的环境和时间发挥它们的作用了，当然这时你的实验也就完了。

下面，我们介绍一个可以确认RNA溶液中有没有残留的RNA酶的方法。

3）保温试验
方法很简单的，按照样品浓度，从RNA溶液中吸取两份1000 ng的RNA加入至0.5 ml的离心管中,并且用pH7.0的Tris缓冲液补充到10 ul的总体积，然后密闭管盖。把其中一份放入70℃的恒温水浴中,保温1 h。另一份放置在-20℃冰箱中保存1 h。

时间到了之后，取出两份样本进行电泳。电泳完成后，比较两者的电泳条带。如果两者的条带一致或者无明显差别（当然，它们的条带也要符合方法2中的条件），则说明RNA溶液中没有残留的RNA酶污染，RNA的质量很好。相反的，如果70℃保温的样本有明显的降解，则说明RNA溶液中有RNA酶污染。

如果你的RNA样本通过了保温实验的检测并且你在后续的实验中还是非常小心的防范RNA酶的骚扰，那么你的实验应该是很难失败了！

实时荧光定量PCR的十大最常见陷阱

引物和探针设计不当
为了最高效地设计用于实时荧光定量PCR的引物和探针，我们强烈建议您使用引物设计软件。大多数引物设计程序均包含了可调节的参数以设计最佳的引物和探针。这些参数涉及了引物/探针的Tm值、互补性、二级结构以及扩增子大小等重要因素。另外还建议您限制连续的重复核酸数目。当设计用于真核目标序列的扩增子时，请选择在目标mRNA中至少跨越一个外显子-外显子接合点的PCR引物对，以避免从残留的基因组DNA模板扩增目标片段。

使用了低质量的RNA
已降解的或低纯度的RNA会抑制反转录效率并降低得率。所用的RNA应当来自于新鲜组织，或者经过RNA稳定试剂处理过的组织，如RNAlater®短片段试剂（70–250 bp）等。这样可以避免少部分降解RNA对结果的影响。若无法使用完全完整的RNA，则可以使设计的引物对与感兴趣基因内部区域进行匹配。需要注意的是，对于真正的实时荧光定量PCR，部分降解的RNA可能无法给出基因表达的精确结果。

未使用“预混液
qRT-PCR是一种分析RNA的高灵敏工具。由于PCR反应会扩增目的基因，所以误差也会被同时扩增出来。因此，无论何时都应当确保变异处于最低水平。“预混液”是反应试剂的混合液，在设置建立多个反应时应当使用它来最小化样品间和反应孔间的差异以提高可重复性。为了进一步减少孔间差异，可以向预混液中加入ROX等参比荧光。

引入交叉污染
对PCR区域的所有表面均应当使用DNA去污染试剂来进行日常去污染工作以防止交叉污染，推荐使用如DNAzap™的试剂，其可以破坏DNA。可以使用“无模板对照“（NTC）来排除试剂和实验桌面的交叉污染。NTC含有除RNA模板外的所有RT-PCR试剂。通常是用无核酸酶水简单地替换掉反应中的RNA。反应后NTC中应当没有合成任何产物；若有产物被合成了，则意味着RT-PCR试剂中的一种或多种被扩增产物污染了。

未使用“– RT”对照
在RNA制备过程中，事实上不可能完全去除基因组DNA。因此，在qRT-PCR实验中有必要加入无逆转录酶对照（“非扩增对照“，还可记为NAC）。通常，NAC是一个模拟的逆转录反应，其中含有除逆转录酶之外的所有RT-PCR试剂。若在NAC中可观察到产物，通常意味着样品中残留有DNA。

使用了不适当的均一化对照
任意qRT-PCR实验的可靠性均可以通过在实验中引入一个不变的内参对照得到改善，这样做有助于改善qRT-PCR的样品间差异以及样品定量过程中的误差。一个好的对照，其表达水平应当在所分析的样品中保持不变。通常我们使用18S rRNA作为对照，因为它相比其他传统内参对照如ß-actin或GAPDH等表达水平变化更小。

使用SYBR® Green时未进行熔解曲线分析
理想情况下，实验样品在扩增子熔解温度下会产生一个清晰的峰（一阶导数图），而NAC及NTC则不会产生任何明显的荧光信号。这类结果意味着PCR产物是特异性的，且此时SYBR Green I 荧光是对目的产物的累积的直接测量。若熔解曲线显示为一系列的峰，则意味着此时很难分辨出特异性及非特异性反应产物。为了获取有意义的数据，这种情况下必须对qRT-PCR进行优化。

没有正确地设置基线和阈值线
为了得到精确的Ct值，应当在丰度最高的样品所对应Ct值两个循环之前设置基线。为了使实时荧光定量PCR的数据有意义，应当在指数扩增期设置阈值线。典型情况下，阈值应当设置在基线至少10个标准差范围内。

扩增效率低
扩增效率(Eff)可以通过以下公式进行计算：
Eff = 10 (–1/slope) – 1
PCR的效率应当在90-110%之间（3.6 > 斜率 > 3.1）。有一系列变量会影响到PCR的效率。举例来说，这些因素包括扩增子的长度、二级结构以及引物设计等。尽管扩增效率落在上述范围之外时，我们也可以得到有效数据，但是仍然应当对qRT-PCR进行进一步优化或改变扩增子设计

使用不正确的范围来制作标准曲线
在RNA定量研究（绝对或相对定量）中或验证PCR（delta-delta-Ct）的扩增效率时，对于每个基因都应当制备标准曲线。标准曲线数据点应当覆盖您的目标基因期望丰度。额外的数据点也可以包括在内，例如在极限上下方的最小及最大RNA量等，以帮助区分特异性和非特异性产物。

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