Biomedical Engineering Reference
In-Depth Information
•
aRNA collection tube (MessageAmp
™
kit component, Ambion)
•
aRNA wash buffer with ethanol added according to manufacturer's directions
(MessageAmp
™
kit component, Ambion)
•
Nuclease-free water (Sigma) preheated to 50
◦
C
•
Vacuum evaporator (SpeedVac; ThermoElectron)
•
UV-V is spectrophotometer.
Method
1 Add 350
μ
l of aRNA binding buffer to an aRNA sample. Mix thoroughly but gently.
2 Add 250
μ
l of 100% (v/v) ethanol.
m
3 Place an aRNA filter cartridge in an aRNA collection tube and transfer the sample
mixture from step 2 onto the center of a filter.
4 Centrifuge at 10 000
g
for 1min.
n
Discard the flow-through and replace the aRNA filter
cartridge in the aRNA collection tube.
5 Add 650
μ
l of aRNA wash buffer to the aRNA filter cartridge.
6 Centrifuge at 10 000
g
for 1min.
n
Discard the flow-through and replace the aRNA filter
cartridge in the aRNA collection tube. Centrifuge at 10 000
g
for an additional 1 min to
ensure that all trace amounts of ethanol are removed.
7 Transfer the filter cartridge to a fresh aRNA collection tube.
8 Add 50
μ
lof50
◦
C (pre-warmed) nuclease-free water
o
to the center of the aRNA filter
cartridge membrane.
9 Incubate at room temperature for 2min and then centrifuge at 10,000
g
for
1.5 min.
10 Repeat steps 8 and 9 with an additional 50
μ
l of nuclease-free water.
11 Determine the aRNA concentration by measuring the absorbance at 260 nm using a
UV-vis spectrophotometer.
12 Aliquot the volume that contains 5
μ
g of aRNA into a new tube, and evaporate the 5
μ
g
sample to dryness using a vacuum centrifugation on low-heat setting, taking care not to
dry the sample excessively.
p
Resuspend the purified sample in 7
μ
l of nuclease-free
water.
Notes
m
Proceed immediately to step 3 after the ethanol is mixed with the aRNA sample, as once this
occurs the aRNA will enter a semi-precipitated state and any delay may result in some loss of
the sample.
n
Additional centrifugation may be required if all the sample does not pass through the filter
after the initial centrifugation. Continue centrifugation until the entire sample has passed
through the filter.
o
Maintain the nuclease-free water at 50
◦
C for the second elution in step 10.
p
The remainder of the sample can be stored at
−
20
◦
C.
