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.