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