Characterization of NGF, trkANGFR, and p75NTR in Retina of Mice Lacking Reelin Glycoprotein
Table 1
Primers for genotyping (A) and for real time PCR (B) used in the study.
Gene access number
Sequence (For/Rev)
Amplicon
Annealing conditions
A: genotyping
Reeler (GM75)
F: 5′-TAA TCT GTC CTC ACT CTG CC-3′
380 bp
55°C, 120 s
Reeler (3W1)
R: 5′-ACA GTT GAC ATA CCT TAA TC-3′
280 bp
Reeler (3R1)
R: 5′-TGT ATT AAT GTG CAG TGT TG-3′
GFP 1
F: 5′-CGT AAA CGG CCA CAA GTT CAG-3′
500 bp
65°C, 30 s
GFP 2
R: 5′-ATG CCG TTC TTC TGC TTG TCG-3′
B: RT-PCR
GAPDH
S: 5′-GTGGACCTCATGGCCTACAT-3′
100 bp
53°C, 30 s
BC059110
AS: 5′-GTTGGGATAGGGACTCCTCAC-3′
trkA
S: 5′-AACAACGGCAACTACAC-3′
137 bp
58°C, 25 s
M23102
AS: 5′-CCTGTTTCTCCGTCCAC-3′
p75NTR
F: 5′-GAGGCACCACCGACAACCTC-3′
131 bp
55°C, 25 s
AF187064
R: 5′-TGCTTGCAGCTGTTCCACCT-3′
NGF
F: 5′-CTGGCCACACTGAGGTGCAT-3′
120 bp
53°C, 30 s
BC011123
R: 5′-TCCTGCAGGGACATTGCTCTC-3′
Amplification profiles: 1 cycle at 94°C/5 min, 30 cycles including 94°C/1 min, 55°C/2 min, and 72°C/3 min, and a final cycle at 72°C/10 min. 1 cycle at 94°C/5 min, 35 cycles including 94°C/30 sec, 65°C/30 sec, and 72°C/30 sec, and a final cycle at 72°C/10 min. 1 cycle at 95°C/15 min, 47 cycles of denaturation at 95°C/30 sec, annealing at 55–60°C/25 sec (primer’s Tm dependent), and elongation at 72°C/30 sec, fluorescence monitoring at 60–90°C, 0.01°C for 0.3 sec, and final incubation at 72°C/5 min. Single melting curves always verified.
