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Prepare injection pads

• line up three 22x50 mm coverslips
• place drop of molten 2% agarose on each
• quickly place slide on top of the agarose and wait 20 seconds (see figure)

• carefully separate coverslips from slide so that the agarose pad remains on the coverslip and dry (overnight at room temp or 15 minutes on 65 degree heat block)
  ** hint: press thumb on coverslips before dripping the agarose - the agarose tends to stick to the "dirty" surface of the coverslip instead of the slide

Load needle with RNA

• pull needles (Narishige puller settings: 4/8/10) from borosilicate glass capillaries
• spin down RNA prep to pellet any debri (full speed 5 minutes)
• load 0.5 ul RNA onto end of needle and allow it to fill the tip by capillary action
• mount needle on micromanipulator
• break needle tip until RNA flows out of the tip

Mount worms for injection

• place drop of heavy mineral oil on pad
• pick up "younger" adult with platinum wire pick
• transfer worm to drop of oil and allow the worm to touch the pad (head or tail)
• upon touching the pad, the worm will begin to stick and you can slide the pick out from under the worm so that the worm lies relatively flat and extended

Inject worm

• set up injector:
  • turn on micro-injector
  • open valve on pressure tank
  • open valve of pressure regulator
  • set injection pressures
    • line pressure: approx 4500 hPa
    • injection pressure: 900 - 1200 hPa
    • back pressure: 80 hPa
• place mounted worm on scope (10X lens) and orient the worm at a 30 degree angle to the needle direction
• switch to higher magnification (40X lens) and bring gonad into focus
  • honeycomb pattern is the surface view - lumen view has granular material in the core
• move needle into the same focal plane
• press needle into the gonad - the gonad will deform around the needle
• tap side of the stage sharply to cause the needle to puncture the gonad wall

• depress peddle to begin filling gonad with solution
• stop filling when gonad expands along entire length but before it "pops" by depressing peddle second time
• pull needle out of gonad
  • clear sticky debri from needle by applying line-pressure to cause solution to flow from tip
• inject other gonad in similar manner

Recover worms

• place drop of egg-salts on worm to "rehydrate" (worm should begin swimming) and allow about five minutes recovery at room temp
• once the worm is swimming well, transfer to OP50-seeded plate with mouth-pipette
• passage worm at regular time intervals to determine timing of onset of RNA-interference effects

Hints

• good needles are the key ...... and largely dependent on your luck when breaking the tip
• break needle by gently pressing tip against 18x18 mm coverslip mounted on drop of oil on 22x50 mm coverslip
• if the worms move too much on the pad try drying the pads on heat block immediately before using
• can do multiple injections on single pad by using small drops of oil at multiple spots: inject worm and recover it in egg salts while mounting and injecting a second worm in neighboring drop, when second is complete, transfer the recovered first to plate, etc ....
• can inject multiple worms within the same drop of oil - just don't do too many that it takes too long and the worms dessicate before you can recover them
• recovery time longer that 5-10 minutes usually not necessary - even non-swimming worms will recover after transfered to seeded plate

References

• Fire, A., et al., (1998) Nature 391:806-811
• Mello, C. C., et al., (1991) EMBO 10:3959-3970

compiled by Chad Rappleye

Aroian Lab Protocols