Mice
euthanized;
hippocampi
dissected
and
snap
frozen
-
2
Total
RNA
preparation;
microRNA
isolation
-
3
RT-PCR
microRNA
relative
expression
Equipment
and
supplies
Dissection
equipment
7900HT
Fast
Real-Time
PCR
System
(Applied
Biosystems,
Foster
City
CA)
Nanodrop
ND-1000
(Thermo
Fisher
Scientific)
Reagents
and
solutions
miRNA
Isolation
Kit
(Ambion,
Life
Technologies,
UK)
TaqMan
RT-PCR
assays
(Applied
Biosystems,
Foster
City
CA)
Primers
for
selected
microRNAs
Procedure:
microRNA
preparation
and
quantification
Mice
are
euthanized
by
cervical
dislocation.
Bilateral
hippocampi
are
dissected
in
their
entirety
from
fresh
brains
within
two
minutes
from
the
time
of
death.
Connective
tissue
is
trimmed
off
and
hippocampi
are
immediately
snap
frozen
on
dry
ice
and
stored
at
-80°C.
Total
RNA
is
isolated
using
a
miRNA
isolation
kit.
RNA
concentration
is
determined
using
a
Nanodrop
ND-1000.
RT-PCR
reactions
are
performed
in
triplicate
using
0.5
µL
of
20x
PCR
Probe/Primer
Mix,
1.5
µL
of
product
from
the
RT
reaction
(diluted
1:10),
5
µL
of
2x
TaqMan
Master
Mix
(no
UNG),
and
3
µL
nuclease-free
water.
A
sample
minus
reverse
transcription
buffer
is
used
as
a
negative
control.
Reactions
are
run
on
a
real
time
PCR
system
in
384-well
format.
RNU19,
miR-9,
and
miR-99a
are
used
as
controls
because
their
expression
does
not
differ
across
strain
and
variability
is
low
within
strain.
Relative
expression
is
calculated
using
the
comparative
Ct
relative
expression
method
in
Microsoft
Excel;
relative
expression
is
normalized
to
the
geometric
mean
of
RNU19,
miR-9,
and
miR-99a.
Definitions
and
calculations
miR
=
miRNA
=
microRNA
Data
collected
by
investigator
microRNA
relative
expression
for
miR-15b,
miR-31,
miR-34c,
miR-212,
miR-201a