Biomedical Engineering Reference
In-Depth Information
2 For each aRNA sample, add 350
μ
l of aRNA binding buffer. Mix thoroughly, but
gently.
3 Add 250
μ
l of 100% (v/v) ethanol.
q
Mix thoroughly, but gently.
4 Place an aRNA filter cartridge in an aRNA collection tube and transfer the sample
mixture from step 2 onto the filter cartridge.
5 Centrifuge at 10 000
g
for 1min.
r
Discard the flow-through and replace the aRNA filter
cartridge in the aRNA collection tube.
6 Add 650
μ
l of aRNA wash buffer to the aRNA filter cartridge.
7 Centrifuge at 10 000
g
for 1min.
r
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.
8 Transfer the filter cartridge to a fresh aRNA collection tube.
9 Add 50
μ
lof50
◦
C (pre-warmed) nuclease-free water
s
to the center of the aRNA filter
cartridge membrane.
10 Incubate at room temperature for 2min and then centrifuge at 10 000
g
for 1.5 min.
11 Repeat steps 8 and 9 with an additional 50
μ
l of nuclease-free water.
t
12 Evaporate each aRNA sample to dryness using a vacuum centrifugation on low-heat
setting, taking care not to dry the sample excessively.
u
Notes
q
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.
r
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.
s
Maintain the nuclease-free water at 50
◦
C for the second elution in step 10.
t
Determine the aRNA concentration by measuring the absorbance at 260 nm using a UV-vis
spectrophotometer (optional).
u
The reason for drying down the sample at this point is to allow for you to resuspend in
the appropriate volume required for your downstream application (hybridization). Following
purification, samples that will be co-hybridized can be combined.
5.2.2 Global-RT-PCR
The global-RT-PCR procedure we present has several advantages. First and foremost, it is
extremely sensitive. We have now used this protocol extensively for the profile of single
cells and have found it to be highly reliable and robust (Figure 5.5). The protocol is also very
quick. T7-based amplification procedures generally take upwards of 2 days to complete; and
when working with very small samples, multiple rounds of IVT are required. By contrast,
the global-RT-PCR procedure can be completed in less than 8 h. Global-RT-PCR results in
DNA-based products, which are more stable, compared with the RNA products produced
as a result of the T7-reaction. In fact, in most cases, the samples can be archived after
