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

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[Bio101] Protocol for knockdown of HuR with siRNA

Cancer Biology > General technique > Biochemical assays > RNA
Author: FengZhi Liu
FengZhi LiuAffiliation: School of Biomedical Sciences, Thomas Jefferson University, Philadelphia, USA
For correspondence: fengzhi6@yahoo.com
Bio-protocol author page: a51
5/20/2012, 6168 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.217

[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: Ambion (Cat.number 4390824 )

2.         Control siRNA (Ambion,Cat. Number. Am4611 ):

3.         OPITMEM (Invitrogen Cat: 3198 )

4.         Oligofectamine (Invitrogen, Cat. 12252-011 )

5.         Culture medium

6.         Water (RNAase free)

 

Equipments

 

1.         6-well plate

2.         Flask T-75

 

Procedure

           

1.        For 6-well plate

1)       Seed about 0.5-0.6X106 to 6-well plate (confluent is 1.2x106) 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, 20μl 1μM of SiRNA (stock 100 μM)or Ctrl SiRNA (stock 50μM) mix with 175 μl of OPTIMEM, stand 5 min. mix 12μl Oligofectamine with 48μl of OPTIMEM, stand 5min.

4)       Mix the two solutions (255μl) and stand 20 min.

5)       Discard media from plate and wash once with OPTIMEM.  Add 250μl of OPTIMEM and the 255μl mixture into well.  Incubate 4-5 Hrs. in 37°C 5% CO2.  After the incubation, add 3ml complete medium (10% FCS DMEM) and 600μl extra FCS (become 15% FCS in the mixture media)

This method usually is for pre experiment. Once you are successful, you can use flask.method.

2.        For flask T-75

1)       Seed cell 2x106 (50% confluent) to T75 flask ahead of one day (30 to 50% confluent is best).

2)       70μl 1μM of SiRNA or Ctrl SiRNA mix with 1225μl of OPTIMEM, 5 min –tube 1

3)       84μl Oligo with 336μl of OPTIMEM, stand 5min-tube 2

4)       Mix the two solutions (1715μl) and stand 20 min at RT.

5)       Discard media from flask and wash one to twicq with OPTIMEM. Add 3.3mL of OPTIMEM into well and mixture 1715μl, total 5 ml. Incubate 4-5 Hrs. After that add 5ml complete medium (10% FCS DMEM) and 1ml 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 wear gloves to take RNA tubes.

You can knockdown other target mRNAs of protein. This is just an example.

 

 



How to cite: Liu, F. (2012). Protocol for knockdown of HuR with siRNA. Bio-protocol Bio101: e217. DOI: 10.21769/BioProtoc.217; Full Text



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11/16/2012 12:14:07 AM  

This post has helped me think things trohguh

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