Preparation of pure total RNA from S.mansoni cercariae

© Julie MJ Lepesant

This protocol is established for extraction of maximum 5000 cercariae.

Material used :

- Glass beads 106 microns and finer (Sigma, cat#G4649)

- 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

• Add a little bit of glass beads in the tube

• Grind the cercariae with a piston in liquid nitrogen for 5 minutes

• Re-suspend the grinded cercariae in 0.5 ml TRIzol® Reagent

• Add 0.1 ml Chloroform per 0.5 ml TRIzol® Reagent used. Cap and shake the tube vigorously by hand for 1 minute

• Incubate at room temperature for 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 10000 rpm for 45 seconds 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 10000 rpm for 45 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 10000 rpm for 45 seconds at room temperature. Discard the flow-through

• Centrifuge at 10000 rpm 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 1 minutes at 8000 rpm at room temperature

• Heat at 65°C for 10 minutes

๏ 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 8000 rpm for 30 seconds at room temperature and discard the flow-through

• Add 500 μl Buffer RPE to the RNeasy spin column

• Centrifuge at 8000 rpm for 30 seconds at room temperature and discard the flow-through

• Add 500 μl Buffer RPE to the RNeasy spin column

• Centrifuge at at 8000 rpm for 30 seconds at room temperature and discard the flow-through

• Place the RNeasy spin colum in a new 2 ml collection tube and centrifuge at at 8000 rpm 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 8000 rpm for 1 minute at room temperature to elute the RNA