Table 1.

Quantification of site-specific methylation

Modified nucleotide	Modified oligonucleotide	HeLa (% modification)a			HeLa (% modification)b		Guide scaRNA (predicted)	Quantitative methods
		ncRNA	siRNA-1	siRNA-2	GFP-ASO	scaRNA-ASO
U1 snRNA
Cap (1Am, 2Um)	TMG-AmUmACYYACCUG	100 ± 0	100 ± 0	100 ± 0			scaRNA16, 18	Peak area
70Am	CAmCUCCG	79.3 ± 3.9	24.2 ± 2.3**	23.4 ± 0.7**	83.9	59.6	scaRNA7	SILNAS
70Am	CAmCUCCG	78.1 ± 5.6	20.3 ± 2.4**	19.2 ± 3.1**	87.7	48.2	scaRNA7	Peak area
U2 snRNA
Cap (1Am, 2Um)	TMG-AmUmC	99.3 ± 0.0	99.1 ± 0.1	99.2 ± 0.1			–	Peak area
11Gm	GmGmC + GmGC	95.3 ± 1.6	92.6 ± 2.7	96.1 ± 0.6	96.9	87.9	scaRNA2	Peak area
12Gm	GmGmC + GGmC	96.2 ± 1.3	93.8 ± 2.3	96.4 ± 0.5			unknown	Peak area
19Gm	UUGmG	76.5 ± 1.4	47.7 ± 2.4*	53.9 ± 4.4*	85.1	57.8	scaRNA9	SILNAS
25Gm	AAGmAU	98.0 ± 1.0	33.9 ± 5.4**	33.1 ± 4.4**	98.3	65.1	scaRNA2	SILNAS
25Gm	AAGmAU	98.7 ± 0.7	35.5 ± 7.4**	29.4 ± 3.5**	98.5	66.3	scaRNA2	Peak area
30Am	Am6AmGU + AAmGU	94.8 ± 1.6	90.9 ± 0.9	93.8 ± 0.6	94.9	86.3	scaRNA9	Peak area
34Ψ	ΨAG	84.0 ± 6.0	79.5 ± 5.4	86.0 ± 6.0			scaRNA8	SILNAS
34Ψ	ΨAG	84.5 ± 6.9	82.5 ± 6.0	82.8 ± 7.8			scaRNA8	Peak area
40Cm	YAYCmYG	95.1 ± 2.5	73.7 ± 2.0**	63.6 ± 0.7**			unknown	Peak area
47Um	YYCUUmAUCAG	95.6 ± 0.8	27.1 ± 1.9**	24.5 ± 1.7**	97.0	54.2	scaRNA28	Peak area
61Cm	UYUAAYAUCmUG	57.2 ± 8.9	20.6 ± 1.4*	16.8 ± 0.6*	65.6	45.3	scaRNA2	Peak area

m; methyl group, Y; U or pseudouridine (Ψ), TMG; 2,2,7-trimethylguanosine (m3G).

a: Values are obtained as mean ± SEM (n = 3). *P < 0.05; **P < 0.01 (Tukey’s test; versus ncRNA). b: Values are given as a single analysis.

TDP-43 mRNA levels (per GAPDH mRNA); ncRNA (1.000 ± 0): siRNA-1 (0.248 ± 0.025): siRNA-2 (0.244 ± 0.013).

In the case of analysis U2-cap, 11Gm, 12Gm and 19Gm; ncRNA (1.000 ± 0), siRNA-1 (0.274 ± 0.025), siRNA-2 (0.288 ± 0.022).