Cell culture materials such as HBSS, L-Glutamine,
0.05% trypsin, and DMEM/F-12 were
purchased from Invitrogen and fetal bovine
serum (FBS) was obtained from Atlas
Biologicals. All other molecular biology
chemicals were purchased from Sigma.
NaB, was acquired from Sigma.
Human Neuroblastoma. Sh-SY5Y
modification were obtained from Sigma,
and were prepared as followed (9,10).
The Institutional Review Board of the
Rush University Medical Center approved
all experimental protocols. About, 8-15
week-old culture cells were used, and had
confluent growth on 24 wells and treatment
for different time frames to create a greater
resolution of RNA production based on
Treatment dilutions of NaB and cinnamic
acid. Time treatments intervals and varying
concentrations were used for NaB and
cinnamic acid. For NaB concentrations
we used a control of zero concentration
then 50µM, 100µM, 150µM, and 200µM;
and time treatment of 30 min, 60min,
120min, 180min, and 240min with 100
µM NaB concentration. For cinnamic acid
concentrations we used a control of zero
concentration then 50µM, 100µM, 200µM,
300µM, and 500µM; and time treatment of
30 min, 60min, 120min, 180min, and
240min with 300 µM cinnamic acid.
RT-PCR and real time qPCR. RNA from
neuroblastomas were isolated using Qiagen
RNA-Easy kit and their protocol’s were
used. cDNA was then synthesized using
dNTP, 0.1M DTT, oligo-dT, RNaseOUT,
M-MLV RT, and 5x FS Buffer all from
Invitrogen which was then amplified with
Promega’s PCR Master Mix with the
primers from Invitrogen.
CREB, (antisense) 5´-TGC CAA GCC AGT CCA
TTC TCC AC-3´; (sense) 5´-TGC AGC TGC CAC
TCA GCC GGG-3´.
BDNF, (antisense) 5´TGT CAC ACA CGC TCA
GCT CCC C-3; (sense) 5´-AGG CAA CTT GGC
CTA CCC AGG TGT G-3´,
NT-3, (antisense) 5´-GTC ATC AAT CCC CCT GCA
ACC GTT T -3´; (sense) 5´-GAG...