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[Bio101] Protocol for knockdown of HuR with siRNA
[Bio101] 利用siRNA技术下调HuR基因的表达

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Abstract

Through a base-pairing-dependent mechanism, 21-23 nucleotide (nt) siRNA binds to complementary target mRNA to inhibit translation.

Materials and Reagents

  1. HuR-siRNA (Life Technologies, Ambion®, catalog number: 4390824 )
  2. Control siRNA (Life Technologies, Ambion®, catalog number: Am4611 )
  3. OPITMEM (Life Technologies, InvitrogenTM, catalog number: 3198 )
  4. Oligofectamine (Life Technologies, InvitrogenTM, catalog number: 12252-011 )
  5. Culture medium
  6. Dulbecco's modified eagle medium (DMEM)
  7. Water (RNase free)

Equipment

  1. 6-well plate
  2. Flask T-75
  3. Gloves

Procedure

  1. For 6-well plate
    1. Seed about 0.5-0.6 x 106 to 6-well plate (confluent is 1.2 x 106) ahead of one day. Culture media is DMEM for this cell line.
    2. About 50% confluent. Ready for tranfection. The following steps are carried out at room temperature (RT). 
    3. For each well in 6-well plate, 20 μl 1 μM of SiRNA (stock 100 μM)or Ctrl SiRNA (stock 50 μM) mix with 175 μl of OPTIMEM, stand 5 min. mix 12 μl Oligofectamine with 48 μl of OPTIMEM, stand 5 min. 
    4. Mix the two solutions (255 μl) and let stand for 20 min.
    5. Discard media from plate and wash once with OPTIMEM. Add 250 μl of OPTIMEM and the 255 μl mixture into well. Incubate 4-5 h. in 37 °C 5% CO2. After the incubation, add 3 ml complete medium (10% FCS DMEM) and 600 μl extra FCS (become 15% FCS in the mixture media).
      This method usually is for pre-experiment. Once you are successful, you can use the flask method.

  2. For flask T-75
    1. Seed cells 2 x 106 (50% confluent) to T-75 flask ahead of one day (30 to 50% confluent is best).
    2. 70 μl 1 μM of SiRNA or Ctrl SiRNA mix with 1,225 μl of OPTIMEM, 5 min –tube 1.
    3. 84 μl Oligo with 336 μl of OPTIMEM, stand 5 min-tube 2.
    4. Mix the two solutions (1,715 μl) and stand 20 min at RT.
    5. Discard media from flask and wash one to twicq with OPTIMEM. Add 3.3 ml of OPTIMEM into well and mixture 1,715 μl, total 5 ml. Incubate 4-5 h. After that add 5 ml complete medium (10% FCS DMEM) and 1 ml FCS (final become 15% FCS in the mixture media). Confirm successful knockdown of HuR in pancreatic cell line by-PCR or by western blot. 

Notes

All siRNA and control RNA should be kept on ice and the experimenter should wear gloves to take RNA tubes. You can knockdown other target mRNAs of proteins. This is just an example.

简介

根据碱基互补配对的原理,通过21-23nt的siRNA与目的mRNA互补配对来抑制翻译的进行。

材料和试剂


 

1.       Apple转换空间"> HuR-siRNA:Ambion   Cat.number 4390824

2.       Apple转换空间"> Control siRNA Ambion -size:10pt; font-family:Arial;"lang ="PT-BR">, ):

3.       Apple转换空间"> OPITMEM Invitrogen Cat:319 8)

4.       Apple转换空间"> Oligofectamine Invitrogen,Cat.12252-011

5.       Apple转换空间"> 培养基

6.       Apple转换空间"> 水(不含RNA酶)

 

设备

 

1.       Apple转换空间"> 6孔板

2.       Apple转换空间"> Flask T-75

 

过程

           

1.      convert-space"> 6孔板

1)       向6孔板种植约0.5-0.6X10 6 文化媒体    是此单元格行的DMEM。

2)       lang ="EN-US">约50%汇合。准备转染。以下步骤在室温(RT)下进行。

3)       对于6孔板中的每个孔,加入20μl1μM的SiRNA(储备100 μM >)或Ctrl SiRNA(库存 50μM)   与175μlOPTIMEM混合,放置5分钟。将12μlOligofectamine与48μlOPTIMEM混合,静置5分钟。

4)       lang ="EN-US">混合两种溶液(255μl)并静置20分钟。

5)         添加250μlOPTIMEM和   孵育4-5小时。   在37°C   5%CO2。孵育后,加入3ml完全培养基(10%FCS DMEM)和600μl额外的FCS(在混合物培养基中变成15%FCS)。/span>

此方法通常用于预实验。一旦成功,您可以使用flask.method。

2.      convert-space"> 对于T-75烧瓶

1)       一天之前,向T75瓶中加入种子细胞2x10 6 (50%汇合) 30至50%汇合是最好的)。

2)       >70μl1μM的SiRNA或Ctrl SiRNA混合物与1225μlOPTIMEM,5分钟管1

3)       用336μlOPTIMEM稀释84μl寡核苷酸,放置5分钟管2

4)       lang ="EN-US">混合两种溶液(1715μl),并在RT放置20分钟

5)       lang ="EN-US">从烧瓶中弃去培养基,用OPTIMEM洗涤一次到twicq。加入3.3mL OPTIMEM加入孔中,加入混合物1715μl,共5ml。孵育4-5小时。然后加入5ml完全培养基(10%FCS DMEM)和1ml FCS(混合物培养基中最终变为15%FCS)。

您可以敲除蛋白质的其他靶mRNA。这只是一个例子。

 

 

  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用:Liu, F. (2012). Protocol for knockdown of HuR with siRNA. Bio-protocol Bio101: e217. DOI: 10.21769/BioProtoc.217;
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This post has helped me think things trohguh
11/16/2012 12:14:07 AM Reply