Growing worms

• on plates
• in liquid

Isolating total RNA

Isolating mRNA

Hybridization

Database Submission/Download

Growing worms:

Large plates:

- Use 150x150mm petri dish plates

- We do not use NGM agar plates for large worm plates.

- Large plates protocol (1.5 L for 20-25 plates):

• 4.5 g NaCl
• 7.5 g Bactopeptone
• 3.75 g Yeast extract
• 30 g Bactoagar
• Add 1.5 L water and stir
• 3 ml cholesterol (5mg/ml in EtOH)

- autoclave (30 min) and let cool for 45 min

- add:

• 1.5 mL 1 M CaCl2
• 1.5 mL 1 M MgSO4
• 37.5 mL 1 M KH2PO4 (pH 6.0)
  

Growing worms on large plates:

Transfer worms to 4 large plates (depending on size of experiment). When worms start to get crowded (but before starving them) cut the agar into 6-8 pieces and transfer each onto a fresh large plate. Remove agar piece one day later. Short before they are running out of food, feed them with E. coli (not OP-50, but fermented, concentrated E. coli; see below: 'Preperation of E. coli in large amounts). One to three days later the worms are ready to harvest (see below: 'Harvesting worms')

Preparation of E. Coli in large amounts

We make our E.Coli for liquid culture worm food in large batches that then are frozen at -80° C and thawed when needed. Lately we have used the DH10B strain. The E. Coli growth is done in a 200L fermenter for several hours, spun down in large centrifuges and has yielded 2-3.5 kg pure bacteria (in paste form). This is then resuspended in a combination of M9 and glycerol until it is a "pipettable" liquid. The following ingredients are needed (based on 200L reaction):

1. 4 L of 5X KH₂PO₄/K₂HPO₄ solution. -----> 462 g KH₂PO₄, 2508 g K₂HPO₄, 4 L H₂O
2. 2.4 kg tryptone
3. 4.8 kg yeast extract
4. 1.6L 50% glycerol
5. 6 L of DH10B starter cultures

Items 2-4 are placed in the fermenter with enough water to fill up tp 200L and then sterilized. The phosphate solution is then sterilely added as are the starter cultures. The bacteria is grown at 37° C for ~4.5 hours and then collected. For resuspending you will need to have autoclaved the following:

1. 4-5L of M9
2. 3 large buckets and spoons

You will also want to mix in some glycerol once resuspended (~20%).

Liquid Culture of Worms (not recommended for expression analysis)

Growing the worms in liquid:

Start by growing 5-10 medium (100 mm) plates of your worms containing a mixture of young and adult worms. When they have nearly starved the plates, wash the worms off with S-basal or M9 solution, pipetting the liquid onto each plate and swirling before pipetting off. Wash 2or 3 times to maximize the number of worms you get. You can wash them directly into 500 mL of S-basal in a baffled flask or if you want to quantitate the volume of worms, collect in a 50 mL conical first (or 15 mL conical) and spin down. Then pipette into your flask. Add 1-3mL E. Coli (amount depends on amount of worms and/or quality of batch of E.Coli but generally speaking start with no more than 1 mL). Shake @20° C on a platform shaker at ~240 rpm. Take approximately 100-200 uL aliquot every day and dump it on an unseeded worm plate to check that the worms are growing and whether they need more food. (It is perhaps easiest to judge food necessity by how clear/opaque the liquid appears after a few minutes of letting the worms settle to the bottom of the flask.) In general, we have found that feeding a culture 1-3 mL a day works well. After a few days you will notice the culture become cluttered with brown oval pellets, presumably worm debris/waste. After 4-5 days (longer for some mutants) your worms should be gravid adults and ready to harvest. If dauers are forming you have probably not added enough food and/or the culture is too dense. However, care must be taken to not add too much food at any one time as the worms can suffocate from lack of oxygen. If after 5 days you do not have enough gravid worms, it is possible to let the culture grow another generation but you will have to clean the culture by sucrose floating (see below) and then resuspend only 0.75-1.0 mL worms per flask.

Once you have gravid adults, you will want to first separate the live worms from all the debris/dead worms in the culture. This can be done by sucrose floating as follows:

1. Prepare ice cold 0.1 M NaCl and ice cold 60% sucrose. (We like to keep 1-1.5L stocks in the cold room)
2. Spin down the worms in 50mL conicals in a clinical centrifuge (highest or 2^nd highest setting; this will take you 2-3 rounds of 4 conicals at a time). Spin time is not that critical, approx. 1 minute is sufficient to yield a soft brown pellet at the bottom of your tubes.
3. Pour off supernatant , resuspend the pellet s in the 0.1 M NaCl and spin to wash, ultimately combining everything into 1 or two tubes depending on your pellet volumes. It is not advisable to have more than 15 mL in each tube.
4. Add NaCl to bring the volume of worms/liquid up to 25 mL in each tube. Mix well to disperse pellet and let sit on ice for > 10minutes. Then add 25mL cold 60% sucrose to each tube, mix, and spin on high for 5 minutes in the clinical centrifuge. The sucrose solution damages and eventually kills the worms so work fast here. Don't let them be exposed to the sucrose for much more than the 5 minutes.
5. After you spin, you should see a brown pellet at the bottom of the tube and a light brown layer of worms floating at the top. Use a broken pasteur pipette tip or a 10mL glass pipette to carefully remove the worms into a new 50 ml conical with approx. 35 mL cold 0.1 M NaCl.
6. Spin the diluted worms for ~1 minute, pour off supernatant and repeat the wash 2-3 more times to get rid of all sucrose.
7. At this point you are ready to collect eggs. To your worm pellet add 2.5 times the volume of hypochlorite solution ( 40 mL 5% bleach, 5 mL 10M NaOH, fill to 100 mL volume with H₂O). Mix gently at room temperature for 5 minutes. This will kill the adults and larvae but not the eggs. It should start disintegrating the carcasses as well though usually not completely. Spin 1 minute and decant supernatant. Wash 3-4 times with 0.1 M NaCl, at some point consolidating into 1 tube. Wash the eggs into 100 mL of fresh S. Basal and let shake O/N at 20° C to hatch.

Note: for doing timecourses you will need to grow up 3-5 cultures of worms rather than 1.

3-4 days before you know you will be bleaching your worms and collecting eggs, you will want to pour ~60 large plates (150mm) with the following media:

1. Add together:

   4.5 g NaCl
   3.75 g Bactopeptone
   30g Bactoagar (Difco)
   7.5 g Bactotryptone
   3.75 g Yeast extract
   3.0 mL cholesterol (5 mg/mL in ethanol)
   1.5 Liter distilled water

2. autoclave 30 minutes

3. mix in, using sterile technique, in the following order:

   1.5 mL CaCl₂ (1M)
   1.5 mL MgSO₄ (1M)
   37.5 mL Potassium Phosphate (1M, pH 6) mix thoroughly

4. pour into plates using sterile technique (~4.5 L = 60 plates). Once they are cooled, spread with E. Coli (not from the frozen down stocks but from a flask of LB broth that has been innoculated with E.Coli) and let grow 2-3 days.

The day after you bleached your worms you will want to plate the eggs onto these plates. We have found it useful to sucrose float once more to separate the L1's from the carcasses from which they hatched. A manageable mount of worms per plate is approx. 100 uL spun-down worms pre-sucrose floating or a proportional amount of sucrose-floated worms (approximately 10-50 ul). Plates are then put at 20° C.

For L3 staging timecourse, you will want to begin monitoring your worms under the Nomarski scope 32-36 hrs after plating. We usually begin collecting 1/7^th of the plates when the Pnp cells are indicative of early L3 stage, that is they are fairly big and look ready to divide for the first time. Time collection will vary depending on the specific experiment you want to do.

Harvesting worms

You will want to keep a large stock of M9 at 20° C in preparation for harvesting. Doing almost everything at 20° C, pipette M9 onto the plates you want to collect (several mLs per plate) and let shake or swirl by hand for a few seconds/minutes . Then pipette off the liquid with worms into 50 ml conicals. Repeat step again and wash into conicals. Spin worms down briefly in clinical centrifuge and wash with M9 2-3 times to get rid of as much bacteria as possible. In the past we have had trouble with bacteria washing off in flakes and then spinning down with the worms, thereby inflating the real volume yield of worms. For 7 plates we usually see ~1 mL worms. Transfer to 50 mL conical and add up to 10ml Trizol per ml packed worms. Vortex briefly, flash freeze in liquid nitrogen, thaw at 37° C and repeat sequence once more. Freeze at -80° C until ready to make RNA.

Isolation of total RNA from worms:

You need:

Trizol (Gibco, # 15596018)
Filter tips and gloves
Isopropanol or Ethanol, 70% EtOH (for RNA work)
DEPC water (or 10mM Tris-CL, pH 7.4, RNAse free)
liquid Nitrogen

Protocol:

1. Wash worms with M9 or S-basal buffer from large plates and pellet them in 50 ml tubes.
   
2. Add Trizol to pellet of packed worms (done right after harvesting). Use up to 10 ml Trizol per ml of packed worms.
   
3. Vortex tubes for 30 sec, then place in liquid Nitrogen, let thaw at 37 deg C, and repeat several times (3-6). Then freeze at -80 deg C until ready to prep.
   
4. Thaw frozen worms/Trizol mixture and vortex 30 sec and ice 30 sec. Repeat this 6-7 times.
   
5. Let stand at RT for 5 min.
   
6. Add 2 ml chloroform per ml packed worms, shake 15 sec by hand, let sit 2-3 min at RT. Parafilm tubes and spin at 4 deg C at 12,000 x g (8250 rpms) for 15 min. RNA is in aqueous supernatant phase. The volume of aqueous is 60% the volume of Trizol used.
   
7. Transfer aqueous phase to a new tube, add 5 ml isopropanol and gently mix (or 2x vol EtOH). Incubate at RT for 10 min (if EtOH: over night at –20 deg C). Centrifugate at 12,000xg (8250 rpm) for 10 min. at 4 deg C. You should see the RNA pellet smeared along the side of the tube.
   
8. Aspirate supernatant, wash RNA pellet with 10 ml 75% EtOH. Centrifuge at 7,500 x g (4000 rpm) for 5 min at 4 deg C.
   
9. Remove all EtOH, but do not dry pellet (is difficult to resuspend). Dissolve in less than 1 ml DEPC water (or 10 mM Tris-Cl, pH 7.4), to resuspend pipet up and down (you can incubate at 55-60 deg C for 10 min). Transfer RNA to an RNAse free Eppendorf tube.
   
10. Run RNA on a gel and take A260 / A280. RNA is pure if ratio is 2.0 (if it is 1.6, the RNA is not completely dissolved or there is protein contamination). Expect 1-4 mg/gram of worms.
    
11. Store frozen at -80 deg C, use only filter tips and use gloved hands when accessing RNA.
    

Isolation of mRNA from worms:

You need:

oligo dT Cellulose: Ambion, Cat.# 10020, 1gm, store at -20 deg C
Biorad columns: Bio-Rad Poly-Prep Chromatography Columns, Cat.# 731-1550, 50 Columns, 0.8x4 cm

Solutions (all RNAse free):

10 mM Tris-Cl, pH 7.4
1x NETS: 100 mM NaCl, 10 mM Tris-Cl pH 7.4, 10 mM EDTA, 0.2% SDS
2x NETS: 200 mM NaCl, 20 mM Tris-Cl pH 7.4, 20 mM EDTA, 0.4% SDS
Isopropanol or Ethanol, 70% EtOH (for RNA work)


Protocol:

• Prepare oligo dT cellulose as follows:
  
  • Resuspend 1g of oligo dT resin in 10 mL 1x NETS
    
  • Wash 3 times with 10 mL 1x NETS
    
  • Resuspend 1g resin in 10 mL 2x NETS for a final volume of ~13 mL (~75 mg/mL)
    
• Dilute 1-2 mg of total RNA to 1 mL with 10 mM Tris-Cl pH 7.4.
  
• In a 10 mL Biorad mini column (sealed on bottom), mix 0.5 mL resin (in 2x NETS) with 1 mL total RNA (1-2 mg, not more than 4 mg). Bind at room temperature on shaker for 1 hr.
  
• Break off bottom of column and mount in 15 mL Falcon tubes.
  
  • Collect:
    
  • flow-through
    
  • washes 5 times with 0.7 mL 1x NETS (all washes into one tube)
    
  • eluates 4 times with 0.7 mL 10 mM Tris-Cl pH 7.4 heated to 70 deg C (eluates into new tube!)
    
  • Note: if cellulose comes through the frit, do a chloroform extraction to get rid of it.
    
• Extract 1-2x with 1x vol of CHCl3 (spin at RT and take the top layer)
  
• Divide eluate into two 1.5 mL eppendorf tubes. Precipitate with 0.1x vol of 3N NaOAc in DEPC H2O, 1x vol of isopropanol at -20 deg C for 1 hour, then spin 30 min at 4 deg C. Wash in 70% EtOH.
  
• Resuspend poly-A RNA in 10-40 uL 10 mM Tris-Cl pH 7.4. Quantitate by A260 and store at -80 deg C. You may also typically run a gel of the RNA eluates to verify that you have a nice smear and that RNA is not degraded.
  

Labeling and Micrarray Hybridization:

Reverse Transcription with fluorescent dye protocol

You need:

Anchored dT oligo: 22-mer: 5'-TTTTTTTTTTTTTTTTTTTTVN-3'
Trimix for dUTP: Total 9.6 uL per reaction; make up stock (ex: x 50 for 480ul stock):

• 0.6 uL of a 25mM dATP, dCTP, dGTP/10mM dTTP mix
  
• 6uL of 5x 1st strand buffer (comes with Reverse Transcriptase enzyme)
  
• 3uL 0.1 M DTT
  

FluoroLink Cy3/Cy5-dUTP (Amersham Pharmacia, Cat.# PA53022/PA55022)
Superscript II Reverse Transcriptase (Invitrogen, Cat.# 18064014)
Ribonuclease Inhibitor (Invitrogen, Cat.# 15518012)
Microcon YM-30 colums (Millipore, Cat.# 42410) or Qiagen PCR Purification Kit (Cat.# 28104)
Yeast tRNA (Invitrogen, Cat.# 15401029)

Protocol Labeling:

one reaction for sample RNA, another reaction for reference RNA (use Cy-3 for reference RNA)

70 degrees for 10 min. Chill on ice for 8 min.

Incubate at 42 degrees for 1.5 hrs.
It is a good idea to spike the reaction with additional enzyme half way through.

Hybridization Protocol (with Micron30):

• Don't put on ice after RT and keep out of light.
  
• Degrade RNA at 65 deg C for 10 min with 1.5 uL 1 M NaOH (no more! Cy5 is sensitive to base and will degrade)
  
• Neutralize with 1.5 uL 1M HCl and quickly dilute with TE to 500 uL.
  
• Spin in micron-30 for 8 min. Dilute again with TE and spin through same column another 8 min.
  
• Invert and collect samples. This is a 2 min spin.
  
• Combine appropriate combinations of probe in single tube and add 1.5 uL carrier DNA (salmon sperm; 2ug/ul or yeast tRNA at 5ug/ul)
  
• Dilute to 500 uL with TE and spin through micron-30 for 3rd time. Collect approx. 10-14 uL probe.
  
• To probe add 4 uL 20x SSC and 0.5uL 10% SDS
  
• Bring total volume of sample up to 27ul.
  
• Heat to 100 deg for 1-2 min.
  
• Cool greater than 10 min, spin/sit at rt. Drop onto array, put cover slip on, place in chamber. Also add 5uL of 3x SSC to edge of slide to keep from drying out.
  
• Hybridize at 65 deg C over night (15-20 hr).
  
  
  

Alternative Hybridization Protocol (less background)(with Qiagen PCR purification kit):

• Don't put on ice after RT and keep out of light.
  
• Degrade RNA at 65 deg C for 10 min with 1.5 uL 1 M NaOH (no more! Cy5 is sensitive to base and will degrade)
  
• Neutralize with 1.5 uL 1M HCl and quickly add 70 uL TE.
  
• Add 500 uL PB buffer (from Qiagen PCR purification kit).
  
• Apply to Qia-Quick column and spin at 13,000 rpm for 30-60 sec.
  
• Aspirate off flow-thru.
  
• Add 750 uL buffer PE and spin again 30-60 sec.
  
• Aspirate off flow-thru and repeat.
  
• Aspirate off flow-thru and spin for 1 min at high speed to dry column.
  
• Transfer to fresh eppendorf.
  
• Add 30 uL buffer EB (or 10mM Tris-Cl, pH 7.4) to center of filter and let sit 1 min at rt.
  
• Spin at 13,000 rpm for 1 min.
  
• (Repeat eluation step)
  
• Dry down Qia-Qick eluate in speed-vac (can take up to 60 min)
  
• Bring volume to 21 uL with water, TE or 10mM Tris-Cl, pH 7.4
  
• Add 1.5 uL carrier DNA (2ug/ul salmon sperm or yeast tRNA at 5ug/ul)
  
• Add 4 uL 20x SSC and 0.5uL 10% SDS
  
• Heat to 100 deg C for 1-2 min.
  
• Cool greater than 10 min, spin/sit at rt. Drop onto array, put cover slip on, place in chamber. Also add 5uL of 3x SSC to edge of slide to keep from drying out.
  
• Hybridize at 65 deg C over night (15-20 hr).
  

Upload of Data into SMD and Download of Data from SMD:

- Upload:

- Download: