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半定量RT-PCR检测草莓基因表达   

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摘要:采用商业化试剂盒提取草莓组织中的总RNA,以其中的mRNA为模板,采用Oligo dT在逆转录酶的作用下反转为cDNA,再以cDNA为模板进行PCR扩增,从而检测目的基因的表达。RT-PCR可对目的基因的表达丰度及基因间表达的差异进行检测,易于操作,周期短。

关键词: 草莓, 基因表达, PCR

材料与试剂

  1. 离心管
  2. Oligo dT18
  3. DEPC-H2O
  4. 2x PCR Taq Master
  5. RRI
  6. MLV
  7. TAE缓冲液
  8. 琼脂糖

仪器设备

  1. 移液枪
  2. 离心机
  3. -20 °C冰箱
  4. PCR仪
  5. 电泳仪

实验步骤

  1. 反转录体系cDNA第一链合成 (25 μl)
    根据测定的浓度计算1 μg总RNA所需的体积
    1)
    2 μl oligodT18 (反转录引物) + 1 μg总RNA + DEPC-H2O = 15 μl,轻匀混合,稍离心;
    2)
    70 °C 10 min;
    3)
    立即冰浴5 min;
    4)
    加混合液10 μl (每管10 μl,根据反应的数量调整相应混合液的量,可适当多配制一些以补偿转移过程中的损失);
    混合液配制 (10 μl):
    M-MLV buffer
    5 μl
    dNTP
    1 μl (浓度10 mM)
    RRI
    0.7 μl
    MLV 1 μl
    DEPC-H2O
    2.3 μl
     注:配置混合液是用枪吸打均匀,吸打15次。
    5)
    42 °C 1 h,70 °C 10 min (恒温PCR);
    6)
    反转录得到mRNA-cDNA的杂交链,-20 °C保存。

  2. 半定量RT反应 (10 μl体系),在PCR管中混合以下溶液:

    轻匀混合,稍离心
    1)
    在PCR仪上按下述程序反应:
    起始变性:95 °C 3min
    按下述条件进行28个循环
    变性:95 °C 30 sec
    退火:55 °C 30 sec (根据实际温度设定退火温度)
    延伸:72 °C 30 sec (1,000 bp/min) 根据实际长度设定时间
    最终延伸:72 °C 5 min
    2)
    配置1%琼脂糖胶 (见溶液配方) (100 ml TAE缓冲液 + 1 g琼脂糖),然后跑琼脂糖电泳,检测PCR产物。

注意事项

  1. 保证基因间PCR产物长度相近,确保在同一延伸时间内扩增目的片段。
  2. 引物长度在20 bp-25 bp范围内,保证产物的特异性。
  3. 引物退火温度在55 °C左右,上下游引物退火温度保持相近。
  4. 引物自身及上下游引物间应不存在二聚体。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:魏灵芝, 毛文文, 李冰冰, 贾文锁. (2018). 半定量RT-PCR检测草莓基因表达. Bio-101: e1010221. DOI: 10.21769/BioProtoc.1010221.
How to cite: Wei, L. Z., Mao, W. W., Li, B. B. and Jia, W. S. (2018). Semi-quantification of Strawberry Gene Expression by RT-PCR. Bio-101: e1010221. DOI: 10.21769/BioProtoc.1010221.
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