9. How to remove DNA contanimation

I. 为什么要去除RNA中的基因组DNA？

• RNA样本中基因组DNA残留对后期基因表达分析的准确性具有很大影响。残留的基因组DNA一方面会影响RNA浓度的精确定量；另一方面，残留的DNA也会影响qRT-PCR扩增效率，及建库过程中的准确性。

• 无论用种方式制备的总RNA，要绝对保证不含基因组DNA是不现实的，目前RNA提取过程中去除基因组DNA最常用的方法就是加入DNase Ⅰ（无RNA 酶）消化。DNase I (RNase-free) 是一种内切酶，可切割单链和双链DNA，降解DNA，又不与DNA反应，以保留RNA。

II. DNase I

• Recombinant DNase I (RNase-free), Takara #2270A

• Inactivition: The enzyme loses its activity reversibly with EDTA, and irreversibly by heat treatment at 80 °C for 10 minutes.

• Methods:

  • 1) Prepare the following reaction mixture.

    Reagent	Volume
    Total RNA	20 ~ 50 μg
    10 × DNase I Buffer (supplied)	5 μl
    Recombinant DNase I (RNase-free)	2 μl (10 units)
    Ribonuclease Inhibitor	20 units
    DEPC-treated water	up to 50 μl

  • 2) Incubatefor20~30min.at37°C.

  • 3) Perform the following Heat treatment procedure to inactivate DNaseI: Add 2.5 μl of 0.5 M EDTA, incubate at 80 °C for 2 min.

  • 4) Following RNA clean & concentration operation.