© Julie Lepesant, 2011

This protocol is established for extraction of maximum 5000 cercariae.

Material used :

• TRIzol® Reagent
• Chloroform
• Ethanol 70% and 100%
• PureLink™ RNA Mini kit (Ambion, cat#12183020)
• TURBO DNA-free™(Ambion, cat#AM1907)
• Rneasy® mini kit (QIAGEN, cat#74104)
• RNase-free water
• RNAsecure (Ambion, cat n°AM7006)

Extraction

• Re-suspend the cercariae pellet (with as few liquid as possible) in 0.5 ml TRIzol® Reagent and transfer the suspension in  a tube containing MagNA Lyser green beads (Roche, cat#03358941001)
• Disintegrate samples using a MagNA Lyser 45 seconds at 6000 rpm
• Transfer all the liquid to a new RNase free tube
• Add 0.1 ml Chloroform per 0.5 ml TRIzol® Reagent used. Cap and shake the tube vigorously by hand for 15 seconds
• Incubate at room temperature for 2–3 minutes
• Centrifuge the sample at ≥12,000 × g for 15 minutes at 4°C
• Transfer the colorless, upper phase containing the RNA to a new RNase-free tube.
• Add an equal volume of 70% ethanol to obtain a final ethanol concentration of 35%. Vortex. 
• Proceed to Binding, Washing, and Elution of the PureLink RNA Mini kit (Ambion, cat#12183020)
• Transfer ≤700 μl of sample to a Spin Cartridge (with a Collection Tube)
• Centrifuge at ≥12,000 × g for 1 minute at room temperature. Discard the flow-through
• Discard the flow-through and the collection Tube and insert the Spin Cartridge into a new collection Tube
• Add 500 μl Wash Buffer II with ethanol to the center of the Spin Cartridge
• Centrifuge at ≥12,000 × g for 15 seconds at room temperature. Discard the flow-through
• Insert the Spin Cartridge into a new collection Tube
• Add 500 μl Wash Buffer II with ethanol to the center of the Spin Cartridge
• Centrifuge at ≥12,000 × g for 15 seconds at room temperature. Discard the flow-through
• Centrifuge at ≥12,000 × g for 1 minute at room temperature to dry the membrane with bound RNA. Discard the flow-through and insert the Spin Cartridge into a Recovery Tube
• Add 30 μl RNAsecure (Ambion, cat n°AM7006) to the center of the Spin Cartridge
• Incubate at room temperature for 1 minute
• Centrifuge the Spin Cartridge with the Recovery Tube for 2 minutes at ≥12,000 × g at room temperature

DNase treatment (TURBO DNA-free™, Ambion, cat n°AM1907)

• Add 3 μl of 10X TURBO DNase Buffer to 30 μl of total RNA in RNAsecure
• Add 3 μl of TURBO DNase (2 Units/μl) and mix gently
• Incubate at 37°C for 30 minutes

DNase inactivation and RNA Clean-up ( Rneasy® mini kit, QIAGEN, cat#74104)

• Adjust the sample to a volume of 100 μl with RNase-free water.
• Add 350 μl Buffer RLT, and mix well
• Add 250 μl ethanol (100%) to the diluted RNA, and mix well by pipetting.
• Transfer immediately the sample to an RNeasy Mini kit spin column placed in a 2 ml collection tube.
• Centrifuge at ≥12,000 × g for 15 sec at room temperature and discard the flow-through
• Add 500 μl Buffer RPE to the RNeasy spin column
• Centrifuge at ≥12,000 × g for 15 sec at room temperature and discard the flow-through
• Add 500 μl Buffer RPE to the RNeasy spin column
• Centrifuge at ≥12,000 × g for 15 sec at room temperature and discard the flow-through
• Place the RNeasy spin colum in a new 2 ml collection tube and centrifuge at ≥12,000 × g for 1 minute at room temperature to dry the membrane
• Place the RNeasy spin colum in a new 1.5 ml tube and Add 30 μl RNase-free water directly to the spin column membrane
• Incubate at room temperature for 1 minute.
• Centrifuge at ≥12,000 × g for 1 minute at room temperature to elute the RNA