赛默飞无氯仿提取RNA解决方案来了！-医疗器械网

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赛默飞无氯仿提取RNA解决方案来了！

来源：赛默飞世尔科技生命科学产品分类：维修保养 2024-03-30 10:15:08 37阅读次数

TRIzol试剂

1977年，科学家 Axel Ullrich 和他的同事们提出使用异硫氰酸胍（Guanidine Thiocyanate）用于 RNA 的分离纯化，至1990 年，优化该方法后，推出商业化的 TRIzol 试剂。

赛默飞TRIzol试剂是一种即用型试剂，用于在1小时内从人类、动物、植物、酵母或细菌来源的细胞和组织样本中提取高质量的总RNA（以及DNA和蛋白质），TRIzol试剂在样本匀浆化时，可以破坏细胞，溶解细胞组分，同时高效的抑制RNA酶的活性，从而维持RNA的完整性。

TRIzol无氯仿解决方案

由于在TRIzol试剂的提取过程中，需要使用氯仿，而氯仿具有挥发性，会对眼睛、呼吸道、皮肤和黏膜造成刺激，因此迫切需要一种无氯仿解决方案。经过赛默飞科学家的不懈探索，发现了1-溴-3-氯丙烷（1-Bromo-3-chloropropane ，BCP）可以替代氯仿，搭配TRIzol试剂提取RNA，接下来，小编就和大家一睹为快——TRIzol无氯仿提取RNA的效果。

操作流程

在样本进行匀浆化时，加入0.2mL氯仿或者0.1mL BCP，进行RNA提取。

图1. 操作流程示意图，详细步骤如下：

加1mL TRIzol试剂，裂解样本，室温孵育5min。
加0.2 mL氯仿或 0.1 mL BCP，振荡混匀，孵育3min。
在4°C下12000×g离心样本15min。
取约500μL上清液至新样本管中。
加等样本体积的异丙醇，颠倒混匀，在4°C下孵育10min。
在4°C下12000×g离心10min，弃上清。
加1mL 75%乙醇，重悬沉淀，短暂涡旋样本，在4°C下7500×g离心5min，弃上清液。此步骤重复一次。
干燥5-10min，加20-50μL RNase-Free水，在55°C的水浴孵育10min。

实验结果

分别使用氯仿和BCP对5mg和20mg的兔的肝脏和脑组织，按照TRIzol试剂的说明书进行RNA提取，每组实验设置3个重复，对得到的RNA产物进行含量、纯度、完整性等进行评估。

RNA含量和纯度

图2. Nanodrop检测RNA的产量和纯度

RNA完整性测定

图3. Agilent 2100测定RNA完整性和计算RIN指标

RT-qPCR分析

图4. 对提取RNA进行RT-qPCR检测内参基因：GAPDH和ACTA₂，说明BCP提取的RNA，与氯仿相比，内参基因表达量无显著差异。

图5. 提取RNA进行Xeno抑制性实验，说明，BCP和氯仿所提取出来的RNA不会扩增抑制后续的RT-qPCR实验，降低假阴性结果的可能性，确保RT-qPCR检测结果是准确和可控的，再次佐证了可以使用BCP搭配TRIzol试剂使用。

小结

在有机法提取RNA的过程中，赛默飞无氯仿提取后的RNA，含量高，纯度好，完整性指数高，可直接做后续的RT-qPCR实验。

TRIzol开学季促销

为了助力科研er顺利开启新学期，赛默飞如火如荼地展开“开学季促销”活动：

2月19日-3月31日期间，TRIzol试剂科研好物惊爆价促销，扫码下方二维码或点击「阅读原文」，了解更多。

TRIzol试剂订购信息

*促销至2024年3月31日

用假 TRIzol 提 RNA 十几次后，怨种我总结了 5 大识别方法

????点击「阅读原文」，了解更多信息

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[2]Declaration of app\common\model\Topic::getList($filter = Array) should be compatible with app\common\model\BaseModel::getList($filter = Array, $sort = Array)
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[2]Declaration of app\common\model\TopicInfoList::getAllList($filter = Array, $limit = 8) should be compatible with app\common\model\BaseModel::getAllList($filter = Array, $with = Array, $limit = 0, $sort = Array, $field = 'a.*', $join = Array)
[2]Declaration of app\common\model\News::getAllList($filter = Array, $limit = 16, $order = Array, $with = Array) should be compatible with app\common\model\BaseModel::getAllList($filter = Array, $with = Array, $limit = 0, $sort = Array, $field = 'a.*', $join = Array)
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[ SQL ] SELECT * FROM `tech_news_comment` WHERE `tech_news_id` = 3157 AND `parent_id` = 0 ORDER BY `id` DESC [ RunTime:0.000185s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `tech_news_comment` WHERE `tech_news_id` = 3157 LIMIT 1 [ RunTime:0.000202s ]
[ SQL ] SELECT * FROM `tech_news` WHERE `is_show` = 1 AND `review_status` = 1 AND `id` IN (3157,3865,3859,3832,3783,3704,3685,3594,3529,3342) ORDER BY field(id,3157,3865,3859,3832,3783,3704,3685,3594,3529,3342) [ RunTime:0.000405s ]
[ SQL ] SELECT * FROM `user_info` WHERE `user_id` IN (378,345,722,530,353,252,197,210) [ RunTime:0.000910s ]
[ SQL ] SHOW COLUMNS FROM `topic` [ RunTime:0.001046s ]
[ SQL ] SELECT `id`,`md5`,`title`,`created_time` FROM `topic` WHERE `review_status` = 1 ORDER BY `id` DESC LIMIT 4 [ RunTime:0.000197s ]
[ SQL ] SHOW COLUMNS FROM `topic_info_list` [ RunTime:0.000892s ]
[ SQL ] SELECT * FROM `topic_info_list` WHERE `topic_id` = 1 ORDER BY `id` DESC LIMIT 2 [ RunTime:0.000201s ]
[ SQL ] SHOW COLUMNS FROM `news` [ RunTime:0.000934s ]
[ SQL ] SELECT * FROM `news` WHERE `id` IN (864,849) [ RunTime:0.000216s ]
[ SQL ] SELECT `id`,`md5`,`title`,`created_time` FROM `tech_news` WHERE `review_status` = 1 AND `is_show` = 1 ORDER BY `id` DESC LIMIT 10 [ RunTime:0.000194s ]
[ SQL ] SHOW COLUMNS FROM `user` [ RunTime:0.000853s ]
[ SQL ] SELECT `a`.* FROM `user` `a` INNER JOIN `tech_news` `b` ON `a`.`id`=`b`.`user_id` GROUP BY `a`.`id` ORDER BY `b`.`id` DESC LIMIT 10 [ RunTime:0.004613s ]
[ SQL ] SELECT * FROM `user_info` WHERE `user_id` IN (592,413,444,224,13,239,598,697,394,285) [ RunTime:0.000798s ]
[ SQL ] SHOW COLUMNS FROM `seo` [ RunTime:0.000820s ]
[ SQL ] SELECT * FROM `seo` WHERE `column_type` = '16' AND `page_type` = 'tech_news' AND `info_id` = 3157 LIMIT 1 [ RunTime:0.000233s ]
[ SQL ] SELECT * FROM `tech_news_belong_category` WHERE `tech_news_id` = 3157 [ RunTime:0.001202s ]
[ SQL ] SELECT * FROM `tech_news_belong_category` WHERE `tech_news_id` = 3865 [ RunTime:0.000890s ]
[ SQL ] SELECT * FROM `tech_news_belong_category` WHERE `tech_news_id` = 3859 [ RunTime:0.000892s ]
[ SQL ] SELECT * FROM `tech_news_belong_category` WHERE `tech_news_id` = 3832 [ RunTime:0.000898s ]
[ SQL ] SELECT * FROM `tech_news_belong_category` WHERE `tech_news_id` = 3783 [ RunTime:0.000889s ]
[ SQL ] SELECT * FROM `tech_news_belong_category` WHERE `tech_news_id` = 3704 [ RunTime:0.000903s ]
[ SQL ] SELECT * FROM `tech_news_belong_category` WHERE `tech_news_id` = 3685 [ RunTime:0.001102s ]
[ SQL ] SELECT * FROM `tech_news_belong_category` WHERE `tech_news_id` = 3594 [ RunTime:0.001251s ]
[ SQL ] SELECT * FROM `tech_news_belong_category` WHERE `tech_news_id` = 3529 [ RunTime:0.000924s ]
[ SQL ] SELECT * FROM `tech_news_belong_category` WHERE `tech_news_id` = 3342 [ RunTime:0.001198s ]
[ SQL ] SELECT * FROM `user` WHERE `id` = 378 LIMIT 1 [ RunTime:0.000201s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `follow_user_id` = 592 LIMIT 1 [ RunTime:0.000232s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `user_id` = 592 LIMIT 1 [ RunTime:0.000165s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `follow_user_id` = 413 LIMIT 1 [ RunTime:0.000199s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `user_id` = 413 LIMIT 1 [ RunTime:0.000165s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `follow_user_id` = 444 LIMIT 1 [ RunTime:0.000361s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `user_id` = 444 LIMIT 1 [ RunTime:0.000157s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `follow_user_id` = 224 LIMIT 1 [ RunTime:0.000141s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `user_id` = 224 LIMIT 1 [ RunTime:0.000149s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `follow_user_id` = 13 LIMIT 1 [ RunTime:0.000143s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `user_id` = 13 LIMIT 1 [ RunTime:0.000135s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `follow_user_id` = 239 LIMIT 1 [ RunTime:0.000137s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `user_id` = 239 LIMIT 1 [ RunTime:0.000121s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `follow_user_id` = 598 LIMIT 1 [ RunTime:0.000142s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `user_id` = 598 LIMIT 1 [ RunTime:0.000122s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `follow_user_id` = 697 LIMIT 1 [ RunTime:0.000127s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `user_id` = 697 LIMIT 1 [ RunTime:0.000131s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `follow_user_id` = 394 LIMIT 1 [ RunTime:0.000123s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `user_id` = 394 LIMIT 1 [ RunTime:0.000204s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `follow_user_id` = 285 LIMIT 1 [ RunTime:0.000139s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `user_follow` WHERE `user_id` = 285 LIMIT 1 [ RunTime:0.000126s ]

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