Quantifying SIRT3 and miR-494 Expression in NPCs

The RNA of NPCs was extracted by applying the TRIzol reagent (Invitrogen) based on the manufacturer’s instructions. The RNA content was determined at a wavelength of 260 nm using a spectrometer. Reverse transcription of 1 μg total RNA was used for synthesizing cDNA, and a reaction volume of 10 μL (4.5 μL diluted cDNA, 0.25 μL primers and 5 μL 2 × SYBR Master Mix) was used for PCR amplification. The cycle threshold was recorded. The target gene expression level was normalized to the GAPDH level, and the miR-494 level was normalized to that of U6. The expression of SIRT3 and miR-494 was calculated using the 2^−ΔΔCt approach. The primers employed are provided in Table 1.Table 1

List of primers employed in RT-PCR

Name	Primer	Sequence	Size
Homo GAPDH	Forward	5′- TCAAGAAGGTGGTGAAGCAGG -3′	115 bp
	Reverse	5′- TCAAAGGTGGAGGAGTGGGT -3′
Homo SIRT3	Forward	5’- CTTACTAGAGTGCGGCGGT-3’	220 bp
	Reverse	5’- ACAGGTCCACTCATCTTCGT-3’
U6	Forward	5 ‘- CGCTTCGGCAGCACATATAC -3’
	Reverse	5 ‘- AAATATGGAACGCTTCACGA -3’
hsa-miR-494	Forward	5 ‘-TGCGCAGGTTGTCCGTGTTGTCT-3 ‘
	Reverse	5′- CCAGTGCAGGGTCCGAGGTATT-3′