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