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RNAi-mediated knocking down of gene activities in the nematode Caenorhabditis elegans by using the Ahringer’s feeding RNAi library
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RNAi-mediated knocking down of gene activities in the nematode Caenorhabditis elegans by using the Ahringer’s feeding RNAi library

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Category
Glycosyltransferases & related proteins
Protocol Name

RNAi-mediated knocking down of gene activities in the nematode Caenorhabditis elegans by using the Ahringer’s feeding RNAi library

Authors
Akiyoshi, Sayaka
Graduate School of Systems Life Sciences, Kyushu University Graduate School

Nomura H., Kazuko
Graduate School of Systems Life Sciences, Kyushu University Graduate School

Nomura, Kazuya *
Graduate School of Systems Life Sciences, Kyushu University Graduate School
*To whom correspondence should be addressed.
KeyWords
Reagents

LB medium

Carbenicillin (100 mg/mL solution)

IPTG (0.2 mM)

C. elegans RNAi library (available from Source BioScience, Nottingham, UK)

Instruments

NGM (Nematode Growth Medium) agar plates

Incubator shaker (37°C)

Incubator for worm culture (for incubation at 15–25°C)

A worm pick (handmade with platinum wire attached to a tip of a glass pipette)

A dissecting microscope (e.g. Olympus SZX-12 or SZX-16 with 1.2 ×PF )

A fluorescence microscope with DIC (Differential Interference Contrast) optics (e.g. Olympus BX51 or Leica DMRXA)

Methods
1.

Checking bacterial clones for RNAi:

1)  Comment 1
2) 

 To eliminate the use of unreliable clones in your experiments, reliability check of the RNAi clones is highly recommended. Try http://biocompute.bmi.ac.cn/CelRNAi/ (Qu et al., 2011) for the reliability check.

Comment 0
2.

Location of the bacterial clones for your desired genes in the library plates:

1) 

 Examine the database of Ahringer’s RNAi library (http://www.lifesciences.sourcebioscience.com/media/381254/C.%20elegans%20Database%202012.xlsx ) and identify the well numbers for the feeding RNAi clones in the library plates (384-well plates). See Fig. 1.

Comment 0
3.

Preparation for Feeding RNAi:

1) 

 Day 1: Defrost Bacteria.

Pick bacteria from the glycerol stock of the bacterial clones of your favorite genes (Replica plates of the Ahringer’s feeding RNAi library stored at −80°C are used.).

Inoculate each of them in 1–3 mL of LB media containing 0.1 mg/mL carbenicillin.

Culture the E. coli for 6–8 h.

Comment 0
2) 

 Store the cultures at 4°C until use.

Comment 0
3) 

 Seed the E. coli onto the NGM agar plates (e.g. 80 μL E. coli culture/35 mm plate, or 250 μL E. coli culture/60 mm plate), and dry the plates in a laminar flow hood under sterile condition.

Incubate the plates at 37°C overnight.

Comment 0
4) 

 Day 2: To induce the double stranded RNA synthesis with 0.2 mM IPTG, cover whole surface of each plate with the IPTG solution, and dry them in a laminar flow hood.

Incubate the plates at 37°C for 4–6 h for dsRNA induction.

Comment 0
4.

Starting Feeding RNAi:

1) 

 Pick one L4 stage worm (this is the P0 worm) and put it onto a dsRNA induced plate prepared above.

Let the worm eat the dsRNA induced E. coli at 25°C. See Comments.

Comment 1
5.

Examination of phenotypes under a dissecting microscope

1) 

 After about 20 h, control P0 worms will grow up to gravid hermaphrodites. In some RNAi experiments, effects of RNAi are obvious in P0 worms. See comments.

Comment 1
2) 

 While observing P0 phenotypes, prepare IPTG induced plates as above, and transfer the P0 worm with a worm pick onto the newly prepared plate after one day.

Comment 1
3) 

 Transfer the P0 worm into freshly prepared RNAi plate twice at one day interval, and examine F1 and P0 phenotypes.

Comment 0
4) 

 Score F1 and P0 phenotypes. F1 Phenotypes are usually most obvious in the second and the third plate.

Comment 0
Figure & Legends

Figure & Legends

 

 

Fig. 1. 

In the above image, the location of the bacterial clone for the gene sqv-5 (T24D1.1) is shown at the end of the line (I-5A05) indicating the bacteria can be found in the I-5 plate(chromosome I , No. 5 plate), A5 well. A screen capture image (by K. Nomura) from the SourceBiosciece “C. elegans Database 2012.xlsx”.

(http://www.lifesciences.sourcebioscience.com/media/381254/C.%20elegans%20Database%202012.xlsx )

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Date of registration:2014-09-11 16:36:02
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