Supplementary Information
Methods
To quantify the editing efficiency at the Q/R site of GluR2 mRNA, nested
PCR for GluR2 was conducted after cDNA synthesis with the following primers: for the
first PCR, hG2F1 (5'-TCTGGTTTTCCTTGGGTGCC-3') and hG2R1
(5'-AGATCCTCAGCACTTTCG-3'); for the nested PCR, hG2F2
(5'-GGTTTTCCTTGGGTGCCTTTAT-3’) and hG2R2
(5'-ATCCTCAGCACTTTCGATGG-3'). We confirmed that these primer pairs were
situated in two distinct exons with an intron between them and did not amplify products
originating from other GluR subunits (data not shown). The PCR products (182 bp)
originating from completely edited GluR2 mRNA were digested into two bands (116 bp,
66 bp) with the restriction enzyme BbvI, whereas the products originating from unedited
GluR2 mRNA were digested into three bands (81 bp, 66 bp, 35 bp). Thus, the editing
efficiency of the partially edited GluR2 mRNA was calculated by the quantitative
analysis of each fragment (116 bp, 81 bp, 66 bp, 35 bp) using a 2100 Bioanalyser
(Agilent Technology). In order to show that the GluR2 mRNA, both edited and unedited,
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was derived from single-neuron tissue and not from glial tissue, we confirmed the
presence of microtubule-associated protein-2 (MAP-2) mRNA, coupled with the
absence of glial fibrillary acidic protein (GFAP) mRNA in each RNA sample by a PCR
method. The PCR primer sets were shown in Supplementary Table 1b.
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Supplementary Table 1a. Profiles of the individuals analysed in this study
Individual
Sex/Age
Disease
(years)
Duration of
Postmortem
illness (years)
delay (hours)
A1
M/79
ALS
2.0
15
A2
M/71
ALS
2.0
12
A3
F/24
ALS
0.7
4.5
A4
M/52
ALS
13.0
3
A5
F/79
ALS
1.5
10
D1
M/65
DRPLA
16
15
D2
F/47
DRPLA
26
812
C1
F/28
Hypovolemia from trauma
─
17
C2
F/31
Hypovolemia from trauma
─
6
C3
M/78
Heart failure, not otherwise specified
─
12
C4
M/57
Hypovolemia from rupture of splenic
─
17
─
28
artery, liver cirrhosis
C5
F/27
Hypovolemia from trauma
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Age, age of patient at death; A1-A5, individuals with amyotrophic lateral sclerosis
(ALS); D1-D2, individuals with dentatorubral-pallidoluysian atrophy (DRPLA); C1-C5,
normal controls with no neurological disorders.
Supplementary Table 1b. Sequences of primers used for PCR
Oligonucleotide sequence
Amplified product length (bp)
MAP-2
198
Forward primer
5’-CCAAGGAGTCTGATTGCAGGA-3’
Reverse primer
5’-CCTCAACCACAGCTCAAATGC-3’
GFAP
182
Forward primer
5’-CTTGCGGTCCCTTCTTACTCAC-3’
Reverse primer
5’-CTCAGTCAAAGCAGAGTGGGTG-3’
MAP-2, microtubule-associated protein-2 (GenBank accession no. NM031846); GFAP,
glial fibrillary acidic protein (GenBank accession no. NM002055).
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