# 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.