Page 69 - 77_01

GUZMÁN-­-ARANGUREN
Y
COLS

negative
control
to
prove
that
silencing
of
the
gene
of
interest
is
due
to
specificity

of
siRNA
and
is
not
due
to
non-­-specific
effects.

Table
1.
Small
interfering
RNAs
(siRNAs)
sequences.

siRNAs
Sense
(5´-­-3´)
Anti-­-sense
(5´-­-3´)

SEQ
1
CCGUAGCCGUGAAGAUGCUTT
AGCAUCUUCACGGCUACGGTG

SEQ
2
CCUGCGUCGUGGAGAACAATT
UUGUUCUCCACGACGCAGGTG

SEQ
3
AGGUGUACAGUGACGCACATT
UGUGCGUCACUGUACACCUTG

Transfection
complexes
were
prepared
in
an
Opti-­-MEM
serum
free
medium

(Invitrogen)
by
mixing
7
µl
of
siPORT
NeoFX
transfection
reagent
(Applied

Biosystems)
and
300
nM
of
scramble/FGFR3
siRNAs.
Inmortalized
human

chondrocytes
carrying
the
achondroplasia
mutation
were
platted
in
a
6
well
plate

after
the
addition
of
transfection
complexes.
After
72h,
RNA
and
protein
were

isolated
to
analyze
knockdown
efficiency.

2.3.
Reverse
Transcriptase-­-PCR

Total
RNA
was
extracted
from
the
achondroplastic
chondrocytes
using
the

Nucleospin
RNA/Protein
Kit
(Macherey-­-Nagel,
Düren,
Germany)
according
to
the

instructions
of
the
manufacturer
and
1
µg
was
reverse
transcribed
using

SuperScript
III
First-­-Strand
synthesis
system
Kit
(Invitrogen).

The
FGFR3-­-specific
primers
(forward
5'-­-
TGCTGAATGCCTCCCACG
-­-3'
and

reverse
5'-­-
CCAGGCTCCACTGCTGATG
-­-3')
that
have
been
previously
described
(11,

12)
were
employed
for
amplification
of
FGFR3.
Glyceraldehyde-­-3
phosphate

dehydrogenase
(GAPDH)
was
used
as
an
internal
control
and
GAPDH-­-specific

primers
(forward
5'-­-
ACCACAGTCCATGCCATCAC
-­-3'
and
reverse
5'-­-

TCCACCACCCTGTTGCTGTA
-­-3')
were
purchased
from
Clontech
(Clontech,

Mountain
View,
CA,
USA).
Reactions
were
performed
using
TITANUM
Taq
DNA

polymerase
(Clontech).
For
FGFR3
amplification,
reactions
conditions
were
94
°C

for
3
minutes,
followed
by
35
cycles
at
94
°C
for
1
minute,
60
°C
for
1
minute,
and

72
°C
for
10
minutes.
The
thermal
cycling
conditions
for
GAPDH
amplification

were
94
°C
for
5
minutes,
followed
by
30
cycles
at
94
°C
for
45
seconds,
60
°C
for

45
seconds,
72
°C
for
2
minutes
and
72
°C
for
7
minutes.

PCR
products
were
electrophoresed
on
1.5%
agarose
gel,
stained
by

ethidium
bromide
and
quantified
using
a
Kodak
GL
200
Imaging
system
and
Kodak

Molecular
Imaging
software
(Kodak,
Rochester,
NY,
USA).
The
values
of
the
ratio
of

FGFR3
to
GAPDH
were
calculated
and
normalized
by
scramble
control
when
the

value
of
scramble
control
was
defined
as
100%.

2.4.
Western
Blot
Analysis

Total
protein
from
achondroplastic
chondrocytes
transfected
with
the

siRNAs
were
isolated
using
the
Nucleospin
RNA/Protein
Kit
(Macherey-­-Nagel)
and

protein
concentration
was
determined
by
the
Protein
Quantification
assay

(Macherey-­-Nagel).

6