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This protocol describes the basic work-flow of expanding nematode culture under lab conditions, which serves for the subsequent preparation of RNA (microarray), protein (IP), and DNA/protein (ChIP).

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[Bio101] Expansion of Worms for Microarray, IP, ChIP and Similar Experiments

Molecular Biology > DNA > Microarray
Author: Peichuan Zhang
Peichuan ZhangAffiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, USA
For correspondence: peichuan.zhang@ucsf.edu
Bio-protocol author page: a11
9/5/2011, 6807 views, 1 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.129

[Abstract] This protocol describes the basic work-flow of expanding nematode culture under lab conditions, which serves for the subsequent preparation of RNA (microarray), protein (IP), and DNA/protein (ChIP).

Materials and Reagents

  1. Plates and culture materials:
    1. High growth (HG) plates
    2. Normal growth (NG) plates  
    3. OP50 or RNAi bacteria culture
    4. LB medium (autoclaved)
      Note: Prepare enough plates (with extra ones to replace contaminated plates).

  2. Other materials:
    1. Antibiotics (carbenicillin, tetracycline)
    2. IPTG
    3. Cholesterol
    4. CaCl2
    5. MgSO4
    6. KPO4
    7. KOH
    8. Sodium hypochlorite
    9. M9 buffer
    10. NP40 buffer
    11. O/N culture
    12. Triton X-100 (Sigma-Aldrich, catalog number: 9002-93-1)
    13. Sodium hypochlorite (Thermo Fisher Scientific, catalog number: SS290-1)
    14. 2x bleach solution (see Recipes)

Equipment

  1. Beckman centrifuge and rotor (Beckman Coulter)
  2. Low-temperature incubator (Thermo Fisher Scientific)
  3. 15-ml conical tubes

Procedure

  1. Before Day 1
    1. Prepare HG plates (10 cm in diameter) and NG plates (15 cm).  Prepare enough medium as described in recipe.  Autoclave, cool down to 60 °C, and then add all the supplements (cholesterol, CaCl2, MgSO4, KPO4, antibiotics, etc). Pour ~15 ml/ 10 cm-plate, ~100 ml/ 15 cm-plate.
      Tip 1. Certain RNAi bacteria do not appear to grow well in the presence of both carbenicillin and tetracycline. In this case, omit tetracycline from the culture and use.

  2. Day 1
    1. Inoculate a single colony of OP50 in 5 ml LB or RNAi bacteria in 5 ml LB (supplemented with 100 μg/ml carbenicillin & 12.5 μg /ml tetracycline) O/N (~16 h), 37 °C, 300 rpm.
      Tip 2. Certain RNAi bacteria do not appear to grow well in the presence of both carbenicillin and tetracycline. In this case, omit tetracycline from the culture and use carbenicillin only to keep the selection pressure. The Tn10 transposon confers tetracycline resistance, which simultaneously knocks out the rnc14 gene that encodes an RNase to enhance the production of dsRNA.
      Tip 3. Prepare a mini-prep of plasmid for the RNAi bacteria and confirm the insert for your gene of interest by PCR (T7 primers) & DNA sequencing (M13 forward primer). The RNAi clones from Julie Ahringer’s lab are based on low copy-number plasmid, so don’t be surprised if the concentration is below detection by spectrophotometry.
    2. Inoculate the O/N bacteria culture in 1 L of LB or LB with antibiotics. Grow O/N (~16 h), 37 °C, 300 rpm.
      Cool the O/N culture on ice (it can be stored in the cold room for a few days). Transfer the culture into centrifuge bottles, spin at 7,000 rpm, 10 min, 4 °C.
      While spinning, make - Make 60 ml NP40 buffer before beginning (30 ml 2x buffer + 6 ml 10% NP40 + 24 ml cold ddH2O - chill on ice!).
      Washing sample in each bottle w/ 250 ml cold H2O. Spin 7,000 rpm 10 min.
      Tip 4. Be extremely careful to avoid any possible contamination to the OP50 culture. Good practice of antiseptic operations is always a must to start your own experiments. Seed a few OP50/NG plates to check contamination. OP50, a uracil auxotroph, should form only a thin layer on NG plate. It is considered to be non-pathogenic to worms. Any weird change of color/smell of your bacteria culture may indicate potential contamination by other types of bacteria.  
    3. Seed OP50 (0.5 ml) on 10-cm HG plates (diameter = 8.5 cm, surface area = 57 cm2). Seed RNA bacteria (1.0 ml) on 15-cm NG /carbenicilin (100 μg/ml)/ IPTG (0.5 mM to 1.0 mM) plates (diameter = 14.0 cm, surface area = 154 cm2).
      Incubate the plates at 37 °C, O/N. Cool down to room temperature before use. Alternatively, put on bench for a few days (adding too much bacteria is not a good idea, since it takes time to dry on the plates).
      Store the plates (seeded with bacteria) in cold room, if not used immediately. Most RNAi bacteria plates can be used within 2 weeks to 1 month (and particularly, for certain potent RNAi clones).
      Tip 5: Typically, 6 to 8 HG plates should provide sufficient numbers of gravid adult animals for hypochlorite synchronization (bleaching) and production of eggs (up to several hundred thousand) for 40 to 50 NG/ carb/ IPTG plates.
      However, different strains could have different development/growth conditions (e.g., mitochondria mutant animals such as clk-1 or isp-1 display slow growth and have less progeny) and optimal conditions should be determined before you start any large-scale experiments.

  3. Day 2
    1. Place two agarose plugs of well-fed animals (e.g., wild-type N2) onto each HG plate.
      Prepare another set on the 2nd day, 3rd day, so on and so forth, to prepare enough biological replicates.

  4. Day 4
    1. When most worms are at d1-d2 stages (it takes ~ 4 to 5 days from starved population), start collection.
    2. Wash worms off from HG plates with M9 containing 0.2% Triton X-100 (0.2 ml per 100 ml). Use glass pipet and 15-ml conical tubes.
    3. Need to wash the plates a few times in order to collect as many gravid animals as possible.
    4. Spin in a bench-top IEC clinical centrifuge at speed 5 (~700 x g), 30 sec.
    5. Remove the supernatant carefully and wash worms w/ M9 buffer, once. Centrifuge again, remove supernatant and leave ~7 ml liquid in the 15-ml conical tube.
    6. Add equal volume of 2x bleach. Agitate/shake the tube slowly yet firmly for ~ 2 to 3 min.
      Tip 6: Optimal incubation time with bleach solution requires a pilot test. The best time to stop is when you see broken carcasses are about to disappear. Remember that you need to collect the eggs by centrifugation for another 30 sec, and during this short period of time, bleach will continue to exert more effects to break the body, and also, causes damage to the eggs. Definitely, avoid over-bleaching!
    7. Start centrifugation for 30 sec to spin down eggs when can still be seen in the tubes. Remove supernatant, and wash immediately with plenty of M9 buffer, at least twice.
    8. Add 10 ml M9 buffer and rock the tube on a rotator at desired temperature (typically, 20 °C), O/N.
      Please refer to Caenorhabditis elegans/Phenotypic assay/Synchronization of worm for further description of hypochlorite synchronization.

  5. Day 5 and beyond
    1. Take a few microliter of M9 and count the numbers of arrested L1 (swimming). Presence of many dead eggs would suggest over-bleaching, which may or may not affect your experiments.
    2. Seed ~6,000 arrest L1 per 15-cm NG/ Carb/ IPTG RNAi plate (or ~2,000 - 2,500 per 10-cm plate).  At least 4,000 animals are required to prepare ≥5 μg of total RNA. You need to concentrate arrested L1 if the density is too low. Otherwise, adding too much M9 buffer on a plate will cause hypoxia.
      Tip 7: Do not add too many animals on a plate. Nematodes love to burrow, and this is one reason why you should not use old plates. One way to solve this problem is to increase the concentration of agar from 2.0% to 3.0%.

  6. Whenever necessary, feed animals with concentrated bacteria that you have prepared before. Start with less arrested L1 (e.g., 3,000 - 5,000) to avoid starvation.
    Tip 7: It takes wild-type animals ~50 - 54 h to come out of arrested L1 and develop to young adult (YA) (w/o eggs). Majority (>90%) of animals should be synchronized as young adults at this point. The time is ~68 - 72 h for daf-2 mutant animals, such as daf-2(e1370), while these animals could never be perfectly synchronized (you will see a mixed population of L3, L4 and YA).
  7. Collect animals at appropriate time points according to your own experiments.
    Tip 8: Compact pellet of ~4,000 YA/d1 adult is ~25 μl in volume. Typically, less than 0.5 ml of worm pellets can be collected from a single 15-cm plate, which can provide enough RNA for at least 200 RT-PCR reactions. We can collect up to several hundred mg of proteins from 10,000 adults.

Recipes

  1. 2x Bleach solution (prepare fresh solution every time before use)
    60 ml sodium hypochlorite
    30 ml 5 N KOH
    10 ml ddH2O

Acknowledgments

This protocol was adapted from work performed by members of the Kenyon lab, including PZ. PZ was supported by a postdoctoral fellowship from the Larry Hillblom Foundation.

References

  1. The Ahringer RNAi library


How to cite: Zhang, P. (2011). Expansion of Worms for Microarray, IP, ChIP and Similar Experiments. Bio-protocol Bio101: e129. DOI: 10.21769/BioProtoc.129; Full Text



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6/1/2012 1:48:17 PM  

Anonymous Lin
Test institute

Basically, I'm trying to perform RNAi and then qRT-PCR.

I'm a little unclear about the volume of RNAi culture required to seed a 15cm RNAi plate. The protocol said grow up a 1L culture, spin down and then wash in 250mL cold H2O. After that do I resuspend in H2O or the NP40 buffer? If it is NP40 buffer, what is this buffer and what does it do? What is the volume that I should resuspend the culture in?

6/1/2012 2:19:58 PM  

Peichuan Zhang (Author)
Department of Biochemistry and Biophysics,University of California

If you refer to the first step of RNAi bacteria prep --- culture a single colony O/N in 1L of LB/Carb 100ug per ml/ Tet 12.5ug per ml (never grow them longer than 14 hours) and then concentrate it by centrifugation. Then wash the RNAi bacteria pellet with LB/Carb/Tet once (it is OK to wash it with sterile H2O), and resuspend the pellet with LB/Carb/Tet (1L to 50ml, 20X concentrated). Store it at 4C and this reservoir will be used as food later. Be careful with potential contamination from bacteria or mold. I have read an article from Michael Petrascheck’s lab about preparing bacteria stock for lifespan assays (JoVE 2011 49 Solis & Petrascheck). They also included anti-fungal in their recipe.

To prepare a 15-cm RNAi plate, you just need to seed ~1.0ml regular RNAi culture (not the concentrated stock). Once you have seeded the worms (less than 6,000 per plate, it’d better to use 2.5% or 3.0% agarose to reduce burrow), remember to feed them with bacteria every other day. Spread ~1.0ml or more concentrated stock onto the plate, and make sure that it will dry soon (you don’t want to add too much liquid onto the surface). I’d say 1 plate of worms is quite enough for qPCR.

BTW, I didn’t use liquid worm culture --- I used solid agar plates instead (you need to be careful with contamination problem, and it’s better to have several extra plates as backup).

6/1/2012 2:23:22 PM  

Anonymous Lin
Test institute

I am wondering whether your RNAi agar plates contain Tet? And to make sure there is no contamination problem, do you just try to be careful or do you use any anti-fungal stuff?

6/1/2012 2:33:22 PM  

Peichuan Zhang (Author)
Department of Biochemistry and Biophysics,University of California

Our stock agar plates are NG/Carb/IPTG (no Tet at all). Some people in the lab prefer not to use plates that already contain IPTG, and they dilute the bacteria ~5X to 10X and then add IPTG to 1.0mM for induction (either in liquid culture, or mix IPTG and diluted bacteria and then seed on the plate). This RNAi method appears to work pretty well.

In my case, I didn’t include the anti-fungal agent as I was not sure whether it would have any lifespan effects. So try to be careful with your bench and clean it up with 70% ethanol before and after use. Keep the plates closer to the burner. Always seed the plates a few days before you start the worm culture, which would allow you to identify contaminations (typically mold) on the plates. This should work just fine. If you happen to see sporadic mold contamination, you can chunk the agar away and fix the hole with fresh 2% agarose.

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