Preparation of pure total RNA from S.mansoni cercariae

© Marion Picard, 2014-07-18

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)

Prepare/reserve in advance:

·      Centrifuge at 4°C

·      Waterbath/drybath at 37°C

·      Drybath at 65°C

 

Extraction

use 2 ml Eppendorf tubes, spin down the larvae and remove liquid

Add a little bit of glass beads into the tube

Grind the cercariae with a white piston in liquid nitrogen for 5 minutes (if the sample is in a 1.5 ml tube, use dark-blue piston)

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

  do at least 20 up and down movements with the piston until obtaining a homogenous viscous liquid and wash the piston with TRIzol.

leave 5 min at RT

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

homogenize by inverting the tube several times

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 (about 400 µl for 1 ml TRIzol) 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, label tube also on tube, not only on lid in case lid flies off in the centrifuge

Add 700 μl Wash Buffer I with ethanol to the center of the Spin Cartridge

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

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 (1.5 ml Eppendorf) for 1 minutes at 8000 rpm at room temperature

Heat at 65°C for 10 minutes in dry bath, if possible with 300 rpm shaking

 

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

put on ice 2 min

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 20 minutes

add 1 µl of 10X TURBO DNase Buffer and  3 μl of TURBO DNase

Incubate at 37°C for 10 minutes

put on ice for 5 min

 

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 by pipeting up and down

Add 250 μl ethanol (100%) to the diluted RNA, and mix well by pipetting. vortex

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