a
b
c
Supplementary Fig. 1. mRNA expression measured by RT-PCR of cell-type specific genes in
astrocytes and neurons isolated by FACS from cerebral hemispheres in vivo from
FVB/NTg(GFAP-GFP)14Mes/J or B6.Cg-Tg(Thy1-YFPH)2Jrs/J mice. Southern blot showing
mRNAs for (a) connexin-30, GFAP, Glt-1 and Fgfr3 (astrocyte markers), (b) Gabra-1, KCC2,
Snap25 and Synaptotagmin 1 (neuron markers) and (c) Connexin-47, Mag, Mog and Mbp
(oligodendrocyte markers). The sizes of the PCR products are listed in Supplementary Table 1.
From the left, the first sample is DNA ladder, the 2nd and 3rd samples neurons, the 4th and 5th
samples astrocytes, and the last one intact brain.(From Fu et al., Neurochem. Int., 2012, in press)
Primers for RT-PCR
Protein
Gene
Connexin-30a
Gjb6
Sequence
Size
Forward
Reverse
5’ GGCTTGGTTTTCAGAGATAG 3’
5’ GAGTTGTGTTACCTGCTGC 3’
369 bp
Gfap
Forward
Reverse
5’ TGCTAGCTACATCGAGAAGG 3’
5’ TCCTCTGTCTCTTGCATGTT 3’
525 bp
Glt-1c
Slc1a2
Forward
Reverse
5’ ATGGCATCAACCGAGGGTG 3’
5’ GCTGGATGCTAAAGCCAGC 3’
339 bp
Fgfr3d
Fgfr3
Forward
Reverse
5’ GTCACTGTACTCAAGACTGCAG 3’
5’ GAAACATTGGCCAGAACAGGAC 3’
449 bp
Gabra-1e
Gabra1
Forward
Reverse
5’ TGCGACCATAGAACCGAAAGA 3’
5’ CAGTCGGTCGATTTTGCTGA 3’
91 bp
KCC2f
Slc12a5
Forward
Reverse
5’ TTCATCAACAGCACGGACAC 3’
5’ CTTCTTCTTTCCGCCCTCAT 3’
186 bp
Snap25f
Snap25
Forward
Reverse
5’ GCAGGGTAACAAACGATGCC 3’
5’ CTTCCCAGCATCTTTGTTGC 3’
211 bp
Syt1
Forward
Reverse
5’ GTGAGTGCCAGTCATCCTGAG 3’
5’ CCTTCATGGTCTTCCCTAAGTC 3’
340 bp
Gjc2
Forward
Reverse
5’ CAGATGAGCAATCCAAGTTCACC 3’
5’ ACTATCTTGAAGACCCAGAAGCG 3’
112 bp
GFAPb
Synaptotagminh
Connexin-47i
Magj
Mag
Forward
Reverse
5’ CACCTCGAGTCGCCTTTGCCATCCTGATT 3’
5’ TCTCCATGGCCTTGACTCGGATTTCTGCATAC 3’
366 bp
Mogk
Mog
Forward
Reverse
5’ AGGAAGGGACATGCAGCCGGAG 3’
5’ CTGCATAGCTGCATGACAACTG 3’
166 bp
Mbpj
Mbp
Forward
Reverse
5’ ATGGCATCACAGAAGAGACC 3’
5’ CATGGGAGATCCAGAGCGGC 3’
375 bp
Supplementary Table 1. Primer sequences used for RT-PCR of cell markers.
a.
b.
c.
d.
e.
f.
g.
h.
i.
j.
k.
from McCulloch et al., 2005. Am. J. Physiol. Renal. Physiol. 289, F1304-F1312.
from Yamaguchi et al., 2003. Environ. Toxicol. Pharmacol. 15, 1-8.
from Utsunomiya-Tate et al., 1997. FEBS. Lett. 416, 312-316.
from Perez-Castro et al., 1995. Genomics 30, 157-162.
from Bailey and Toth, 2004. J. Neurosci. 24, 6343-6351.
from Zhu et al., 2008. Epilepsy Res. 79, 201-212.
from Ikebuchi et al., 1998. Am. J. Physiol. 274, C1496-C1500.
From Kwon et al., 1995. Mamm. Genome. 6, 880-881.
from Odermatt et al., 2003. J. Neurosci. 23, 4549-4559.
from Traka et al., 2008. J. Neurosci. 28, 11537-11549.
from Delarasse et al., 2003. J. Clin. Invest. 112, 544-553.
Supplementary Methods
For determination of mRNA expression by reverse transcription polymerase chain reaction
(RT-PCR) cell suspension was prepared by collecting cells in Trizol. RT was performed as
previously described by Li et al. (2008).
PCR amplification was performed in a Robocycler thermocycler with 0.2 M of forward and
reverse nucleotide sequences (primers) and 0.375 units of Taq-polymerase. Initially the template
was denatured by heating to 94oC for 2 min, followed by 40 amplification cycles for Connexin-30,
GFAP, Glt-1, Fgfr3, Gabra-1, KCC2, Snap25, synaptotagmin, Connexin-47, Mag, Mog and Mbp.
Each cycle consisted of three periods, the first for 45 sec at 94oC; the second for 45 sec at 55.4°C
for Connexin-30, GFAP, Glut-1, Gabra-1, Synaptotagmin 1 and Mog, at 56°C for Mbp and Fgfr3,
and at 63°C for Snap25, KCC2, Connexin-47 and Mag; and the third for 90 sec at 72oC. The final
step was extension at 72oC for 10 min. The PCR products were separated by 1% agarose gel
electrophoresis, and captured by Fluorchem 5500 (Alpha Innotech Corporation, San Leandro, CA,
USA).