Superscript 3 reverse transcriptase reagent kit

To analyze the expression level of autophagy-related genes, total RNA was extracted using Novel Plant Total RNA Mini Kit (Novelgene, Taipei, Taiwan) and reverse transcribed using SuperScript III Reverse Transcriptase reagent kit (Thermo Fisher Scientific, Waltham, MA, United States) as per the manufacturer’s instructions. Quantitative PCR was performed on a 7500 Fast-Real Time PCR system (Applied Biosystems, Grand Island, NY, United States) and data were processed using 7500 Software v.2.3. Delta-delta Ct method was used to quantify the expression of examined genes. The housekeeping gene GAPDH was used as an internal control. Primer sequences: ATG5 (5′-CCAGTTTTGGGCCATCAATC-3′ and 5′-AGTGTGTGCAACTGTCCATCTG-3′), ATG13 (5′-AGCAGTGGCAATACCCATGA-3′ and 5′-GCATCAAACTCGCGGACATT-3′), LC3B (5′-GCAGCATCCAACCAAAATCC-3′ and 5′-TCCGTTCACCAACAGGAAGA-3′), BECN1 (5′-CTGTGGAAAAGAACCGCAAGA-3′ and 5′-GGGCATAACGCATCTGGTTT-3′), LAMP-2 (5′-CTGTGCGGTCTTATGCATTG-3′ and 5′-TCATCCCCACAAATGCTTCCT-3′), and GAPDH (5′-TCGGAGTCAACGGATTTGGT-3′ and 5′-ATTTGCCATGGGTGGAATCA-3′) were used.

Total RNAs were extracted from the sage leaves using a RNeasy Plant Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. RNA quality and concentrations were determined using 1.0% agarose gel electrophoresis and analysis by a NanoDrop 2000/2000c Spectrophotometer (Wilmington, DE 19,810 U.S.A.). Moreover, cDNA was synthesized with a SuperScript III Reverse Transcriptase reagent kit (Thermo Fisher Scientific, USA).