{"id":10678,"date":"2025-07-14T00:24:20","date_gmt":"2025-07-13T16:24:20","guid":{"rendered":"https:\/\/fanyiming.life\/?p=10678"},"modified":"2025-12-21T01:03:24","modified_gmt":"2025-12-20T17:03:24","slug":"%e5%8f%8d%e8%bd%ac%e5%bd%95protocol","status":"publish","type":"post","link":"https:\/\/fanyiming.life\/?p=10678","title":{"rendered":"\u53cd\u8f6c\u5f55Protocol"},"content":{"rendered":"\n

\u597d\u7684\uff0c\u8fd9\u662f\u4ece\u60a8\u63d0\u4f9b\u7684\u6587\u4ef6\u4e2d\u63d0\u53d6\u5e76\u7ffb\u8bd1\u7684HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (R312)<\/strong> \u4e2d\u6587\u7248\u5b9e\u9a8c\u6d41\u7a0b\u3002<\/p>\n\n\n\n

\n\n\n\n

\u4ea7\u54c1\u63cf\u8ff0<\/strong><\/h3>\n\n\n\n

HiScript\u00ae III 1st Strand cDNA Synthesis Kit (+gDNA wiper) \u662f HiScript\u00ae II 1st Strand cDNA Synthesis Kit (+gDNA wiper) \u7684\u5347\u7ea7\u7248\uff0c\u5305\u542b\u65b0\u4e00\u4ee3\u9006\u8f6c\u5f55\u9176 HiScript\u00ae III Reverse Transcriptase \u53ca\u4e3a\u5176\u4f18\u5316\u7684 Buffer\uff0c\u8fdb\u4e00\u6b65\u63d0\u5347\u4e86\u7b2c\u4e00\u94fe\u5408\u6210\u7684\u6548\u7387\u3002\u8bd5\u5242\u76d2\u4e2d\u7684 5 \u00d7 gDNA wiper Mix \u53ef\u57282\u5206\u949f\u5185\u5feb\u901f\u53bb\u9664\u57fa\u56e0\u7ec4DNA\u6c61\u67d3\uff0c\u4f7f\u7ed3\u679c\u66f4\u53ef\u9760\uff0c\u5e76\u7b80\u5316\u4e86qPCR\u5f15\u7269\u8bbe\u8ba1\u8fc7\u7a0b\uff0c\u65e0\u9700\u8bbe\u8ba1\u8de8\u8d8a\u5916\u663e\u5b50-\u5916\u663e\u5b50\u8fde\u63a5\u7684\u5f15\u7269\u3002\u8bd5\u5242\u76d2\u5305\u542b\u5355\u7ec4\u5206\u7684\u9006\u8f6c\u5f55\u5f15\u7269 Oligo (dT)20VN \u548c Random hexamers\uff0c\u7528\u6237\u53ef\u6839\u636e\u9700\u8981\u7075\u6d3b\u9009\u62e9\u5f15\u7269\u7528\u4e8e\u540e\u7eed\u5b9e\u9a8c\u3002\u672c\u8bd5\u5242\u76d2\u53ef\u7528\u4e8e\u5408\u6210\u5168\u957fcDNA\uff08\u6700\u957f\u53ef\u8fbe20 kb\uff09\u7528\u4e8e\u514b\u9686\u7b49\u4e0b\u6e38\u5b9e\u9a8c\uff0c\u4e5f\u53ef\u7528\u4e8e\u5408\u6210\u9ad8\u5ea6\u5747\u4e00\u7684cDNA\u7528\u4e8eqPCR\u3002<\/p>\n\n\n\n

\u50a8\u5b58\u6761\u4ef6<\/strong><\/h3>\n\n\n\n

\u5728-30 ~ -15\u00b0C\u4e0b\u50a8\u5b58\uff0c\u5e76\u5728\u22640\u00b0C\u4e0b\u8fd0\u8f93\u3002
\u6ce8\u610f\uff1a10 \u00d7 RT Mix \u542b\u6709\u9ad8\u6d53\u5ea6\u7684DTT\uff0c\u5728\u4f4e\u6e29\u4e0b\u53ef\u80fd\u4f1a\u6790\u51fa\u3002\u4f7f\u7528\u524d\u8bf7\u6062\u590d\u81f3\u5ba4\u6e29\uff0c\u8f7b\u67d4\u6447\u6643\u5e76\u5145\u5206\u6df7\u5300\uff0c\u5f85\u6c89\u6dc0\u5b8c\u5168\u6eb6\u89e3\u540e\u518d\u4f7f\u7528\u3002<\/em><\/p>\n\n\n\n

\u6ce8\u610f\u4e8b\u9879<\/strong><\/h3>\n\n\n\n
    \n
  • \u9632\u6b62RNase\u6c61\u67d3<\/strong>
    \u8bf7\u4fdd\u6301\u5b9e\u9a8c\u533a\u57df\u6e05\u6d01\uff1b\u4f69\u6234\u4e00\u6b21\u6027\u624b\u5957\u548c\u53e3\u7f69\uff1b\u4f7f\u7528\u65e0RNase\u7684\u8017\u6750\uff0c\u5982\u79bb\u5fc3\u7ba1\u548c\u79fb\u6db2\u5668\u5438\u5934\u3002<\/li>\n\n\n\n
  • \u5f15\u7269\u9009\u62e9<\/strong>\n
      \n
    • \u5bf9\u4e8e\u771f\u6838\u751f\u7269RNA\u6a21\u677f\uff0c\u4f7f\u7528Oligo dT\u5f15\u7269\u4e0e\u771f\u6838mRNA\u76843′ Poly A\u5c3e\u914d\u5bf9\uff0c\u53ef\u83b7\u5f97\u6700\u9ad8\u4ea7\u91cf\u7684\u5168\u957fcDNA\u3002<\/li>\n\n\n\n
    • \u57fa\u56e0\u7279\u5f02\u6027\u5f15\u7269\uff08GSP\uff09\u5177\u6709\u6700\u9ad8\u7684\u7279\u5f02\u6027\u3002\u5982\u679cGSP\u5728\u7b2c\u4e00\u94fecDNA\u5408\u6210\u4e2d\u5931\u8d25\uff0c\u53ef\u4f7f\u7528Oligo (dT)20VN\u6216Random hexamers\u8fdb\u884c\u9006\u8f6c\u5f55\u3002<\/li>\n\n\n\n
    • Random hexamers\u7684\u7279\u5f02\u6027\u6700\u4f4e\u3002\u6240\u6709RNA\uff0c\u5305\u62ecmRNA\u3001rRNA\u548ctRNA\uff0c\u90fd\u53ef\u4ee5\u4f5c\u4e3aRandom hexamers\u7684\u6a21\u677f\u3002\u5f53\u76ee\u6807\u533a\u57df\u5177\u6709\u590d\u6742\u7684\u4e8c\u7ea7\u7ed3\u6784\u548c\u9ad8GC\u542b\u91cf\uff0c\u6216\u6a21\u677f\u662f\u539f\u6838\u6765\u6e90\u65f6\uff0c\u82e5Oligo (dT)20VN\u6216GSP\u65e0\u6cd5\u6709\u6548\u5f15\u5bfccDNA\u5408\u6210\uff0c\u53ef\u4f7f\u7528Random hexamers\u4f5c\u4e3a\u5f15\u7269\u3002<\/li>\n\n\n\n
    • \u5bf9\u4e8eqPCR\uff0c\u5efa\u8bae\u5c06Oligo (dT)20VN\u4e0eRandom hexamers\u6309\u63a8\u8350\u6bd4\u4f8b\u6df7\u5408\u4f7f\u7528\uff0c\u53ef\u4f7fmRNA\u5404\u533a\u57df\u7684cDNA\u5408\u6210\u6548\u7387\u76f8\u540c\uff0c\u6709\u52a9\u4e8e\u63d0\u9ad8\u5b9a\u91cf\u7ed3\u679c\u7684\u771f\u5b9e\u6027\u548c\u53ef\u91cd\u590d\u6027\u3002<\/li>\n<\/ul>\n<\/li>\n\n\n\n
    • \u5173\u4e8e\u53bb\u9664\u57fa\u56e0\u7ec4<\/strong>
      \u4e5f\u53ef\u4ee5\u4e0d\u8fdb\u884c\u57fa\u56e0\u7ec4\u53bb\u9664\u6b65\u9aa4\u76f4\u63a5\u8fdb\u884c\u9006\u8f6c\u5f55\uff0c\u7a7a\u4f59\u4f53\u79ef\u7528RNase-free ddH2O\u8865\u8db3\u3002<\/li>\n<\/ul>\n\n\n\n
      \n\n\n\n

      \u5b9e\u9a8c\u6d41\u7a0b<\/strong><\/h3>\n\n\n\n

      \u25c7 \u7528\u4e8e PCR<\/strong><\/h4>\n\n\n\n

      1. RNA \u53d8\u6027<\/strong>*
      \u5728\u65e0RNase\u7684\u79bb\u5fc3\u7ba1\u4e2d\u6df7\u5408\u4ee5\u4e0b\u7ec4\u5206\uff1a<\/p>\n\n\n\n

      \u7ec4\u5206<\/th>\u4f53\u79ef\/\u7528\u91cf<\/th><\/tr><\/thead>
      \u603bRNA \u6216 Poly A+ RNA<\/td>10 pg – 5 \u03bcg \u6216 10 pg – 500 ng<\/td><\/tr>
      RNase-free ddH\u2082O<\/td>\u8865\u81f3 8 \u03bcl<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n

      \u5728 65\u00b0C \u5b75\u80b2 5 \u5206\u949f<\/strong>\uff0c\u7136\u540e\u7acb\u5373\u51b0\u6d74 2 \u5206\u949f<\/strong>\u3002
      *\u53d8\u6027\u6b65\u9aa4\u6709\u52a9\u4e8e\u6253\u5f00\u4e8c\u7ea7\u7ed3\u6784\uff0c\u63d0\u9ad8\u7b2c\u4e00\u94fecDNA\u7684\u4ea7\u91cf\u3002\u5bf9\u4e8e\u957f\u5ea6\u8d85\u8fc73 kb\u7684cDNA\u7247\u6bb5\uff0c\u8bf7\u4e0d\u8981\u5ffd\u7565\u6b64\u53d8\u6027\u6b65\u9aa4\u3002<\/em><\/p>\n\n\n\n

      2. \u53bb\u9664\u57fa\u56e0\u7ec4DNA<\/strong>
      \u5728\u4e0a\u8ff0\u6df7\u5408\u7269\u4e2d\u52a0\u5165\uff1a<\/p>\n\n\n\n

      \u7ec4\u5206<\/th>\u4f53\u79ef<\/th><\/tr><\/thead>
      \u7b2c1\u6b65\u7684\u6df7\u5408\u7269<\/td>8 \u03bcl<\/td><\/tr>
      5 \u00d7 gDNA wiper Mix<\/td>2 \u03bcl<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n

      \u8f7b\u67d4\u5730\u4e0a\u4e0b\u5439\u6253\u6570\u6b21\u4ee5\u5145\u5206\u6df7\u5300\u3002\u5728 42\u00b0C \u5b75\u80b2 2 \u5206\u949f<\/strong>\u3002<\/p>\n\n\n\n

      3. \u7b2c\u4e00\u94fecDNA\u5408\u6210\u53cd\u5e94\u6db2\u914d\u5236<\/strong>
      \u5728\u4e0a\u8ff0\u6df7\u5408\u7269\u4e2d\u52a0\u5165\u4ee5\u4e0b\u7ec4\u5206\uff1a<\/p>\n\n\n\n

      \u7ec4\u5206<\/th>\u4f53\u79ef<\/th><\/tr><\/thead>
      \u7b2c2\u6b65\u7684\u6df7\u5408\u7269<\/td>10 \u03bcl<\/td><\/tr>
      10 \u00d7 RT Mix<\/td>2 \u03bcl<\/td><\/tr>
      HiScript III Enzyme Mix<\/td>2 \u03bcl<\/td><\/tr>
      Oligo (dT)\u2082\u2080VN \u6216 Random hexamers<\/td>1 \u03bcl<\/td><\/tr>
      RNase-free ddH\u2082O<\/td>5 \u03bcl<\/td><\/tr>
      \u603b\u4f53\u7cfb<\/strong><\/td>20 \u03bcl<\/strong><\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n

      \u8f7b\u67d4\u5730\u4e0a\u4e0b\u5439\u6253\u6570\u6b21\u4ee5\u5145\u5206\u6df7\u5300\u3002
      \u25b2\u672c\u4ea7\u54c1\u4e5f\u9002\u7528\u4e8e\u4f7f\u7528GSP\u8fdb\u884c\u9006\u8f6c\u5f55\u3002\u4e3a\u907f\u514dgDNA wiper\u5bf9GSP\u7684\u6f5c\u5728\u5f71\u54cd\uff0c\u8bf7\u5c06GSP (2 pmol)\u52a0\u5165\u6b64\u6df7\u5408\u7269\u4e2d\u3002<\/em><\/p>\n\n\n\n

      4. \u53cd\u5e94\u7a0b\u5e8f<\/strong><\/p>\n\n\n\n

      \u6e29\u5ea6<\/th>\u65f6\u95f4<\/th><\/tr><\/thead>
      25\u00b0C<\/td>5 \u5206\u949f \u1d43<\/td><\/tr>
      37\u00b0C<\/td>45 \u5206\u949f \u1d47<\/td><\/tr>
      85\u00b0C<\/td>5 \u79d2<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n

      \u1d43. \u4ec5\u5728\u4f7f\u7528Random hexamers\u65f6\u9700\u8981\u6b64\u6b65\u9aa4\u3002\u4f7f\u7528Oligo (dT)\u2082\u2080VN\u6216GSP\u65f6\u8bf7\u8df3\u8fc7\u6b64\u6b65\u9aa4\u3002
      \u1d47. \u5bf9\u4e8e\u5177\u6709\u590d\u6742\u4e8c\u7ea7\u7ed3\u6784\u6216\u9ad8GC\u542b\u91cf\u7684\u6a21\u677f\uff0c\u6e29\u5ea6\u53ef\u63d0\u9ad8\u81f350\u00b0C\uff0c\u8fd9\u5c06\u6709\u52a9\u4e8e\u63d0\u9ad8\u4ea7\u91cf\u3002<\/p>\n\n\n\n

      \u4ea7\u7269\u53ef\u7acb\u5373\u7528\u4e8ePCR\uff0c\u6216\u5728-20\u00b0C\u4e0b\u4fdd\u5b586\u4e2a\u6708\u3002\u5efa\u8bae\u5206\u88c5\u540e\u5728-70\u00b0C\u957f\u671f\u4fdd\u5b58\u3002cDNA\u5e94\u907f\u514d\u53cd\u590d\u51bb\u878d\u3002<\/em><\/p>\n\n\n\n

      \n\n\n\n

      \u25c7 \u7528\u4e8e qPCR<\/strong><\/h4>\n\n\n\n

      1. \u53bb\u9664\u57fa\u56e0\u7ec4DNA<\/strong>
      \u5728\u65e0RNase\u7684\u79bb\u5fc3\u7ba1\u4e2d\u6df7\u5408\u4ee5\u4e0b\u7ec4\u5206\uff1a<\/p>\n\n\n\n

      \u7ec4\u5206<\/th>\u4f53\u79ef\/\u7528\u91cf<\/th><\/tr><\/thead>
      \u603bRNA \u6216 Poly A+ RNA<\/td>10 pg – 1 \u03bcg \u6216 10 pg – 100 ng<\/td><\/tr>
      5 \u00d7 gDNA wiper Mix<\/td>2 \u03bcl<\/td><\/tr>
      RNase-free ddH\u2082O<\/td>\u8865\u81f3 10 \u03bcl<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n

      \u8f7b\u67d4\u5730\u4e0a\u4e0b\u5439\u6253\u6570\u6b21\u4ee5\u5145\u5206\u6df7\u5300\u3002\u5728 42\u00b0C \u5b75\u80b2 2 \u5206\u949f<\/strong>\u3002<\/p>\n\n\n\n

      2. \u7b2c\u4e00\u94fecDNA\u5408\u6210\u53cd\u5e94\u6db2\u914d\u5236<\/strong>
      \u5728\u4e0a\u8ff0\u6df7\u5408\u7269\u4e2d\u52a0\u5165\u4ee5\u4e0b\u7ec4\u5206\uff1a<\/p>\n\n\n\n

      \u7ec4\u5206<\/th>\u4f53\u79ef<\/th><\/tr><\/thead>
      \u7b2c1\u6b65\u7684\u6df7\u5408\u7269<\/td>10 \u03bcl<\/td><\/tr>
      10 \u00d7 RT Mix<\/td>2 \u03bcl<\/td><\/tr>
      HiScript III Enzyme Mix<\/td>2 \u03bcl<\/td><\/tr>
      Oligo (dT)\u2082\u2080VN<\/td>1 \u03bcl<\/td><\/tr>
      Random hexamers<\/td>1 \u03bcl<\/td><\/tr>
      RNase-free ddH\u2082O<\/td>4 \u03bcl<\/td><\/tr>
      \u603b\u4f53\u7cfb<\/strong><\/td>20 \u03bcl<\/strong><\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n

      \u8f7b\u67d4\u5730\u4e0a\u4e0b\u5439\u6253\u6570\u6b21\u4ee5\u5145\u5206\u6df7\u5300\u3002
      \u25b2\u672c\u4ea7\u54c1\u4e5f\u9002\u7528\u4e8e\u4f7f\u7528GSP\u8fdb\u884c\u9006\u8f6c\u5f55\u3002\u4e3a\u907f\u514dgDNA wiper\u5bf9GSP\u7684\u6f5c\u5728\u5f71\u54cd\uff0c\u8bf7\u5c06GSP (2 pmol)\u52a0\u5165\u6b64\u6df7\u5408\u7269\u4e2d\u3002<\/em><\/p>\n\n\n\n

      3. \u53cd\u5e94\u7a0b\u5e8f<\/strong><\/p>\n\n\n\n

      \u6e29\u5ea6<\/th>\u65f6\u95f4<\/th><\/tr><\/thead>
      37\u00b0C*<\/td>15 \u5206\u949f<\/td><\/tr>
      85\u00b0C<\/td>5 \u79d2<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n

      *\u5bf9\u4e8e\u5177\u6709\u590d\u6742\u4e8c\u7ea7\u7ed3\u6784\u6216\u9ad8GC\u542b\u91cf\u7684\u6a21\u677f\uff0c\u6e29\u5ea6\u53ef\u63d0\u9ad8\u81f350\u00b0C\uff0c\u8fd9\u5c06\u6709\u52a9\u4e8e\u63d0\u9ad8\u4ea7\u91cf\u3002<\/em><\/p>\n\n\n\n

      \u4ea7\u7269\u53ef\u7acb\u5373\u7528\u4e8eqPCR\uff0c\u6216\u5728-20\u00b0C\u4e0b\u4fdd\u5b586\u4e2a\u6708\u3002\u5efa\u8bae\u5206\u88c5\u540e\u5728-70\u00b0C\u957f\u671f\u4fdd\u5b58\u3002cDNA\u5e94\u907f\u514d\u53cd\u590d\u51bb\u878d\u3002<\/em><\/p>\n","protected":false},"excerpt":{"rendered":"

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