Abi 7500 ht real time pcr machine

Total RNA was extracted from follicles using a Trizol reagent (Vazyme, Nanjing, China). RNA concentrations were measured using a NanoDrop 2000c (Thermo Scientific, Waltham, USA). The cDNA was generated from 2 μg total RNA using a HiScript II 1^st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) according to the manufacturer's protocol. qPCR was performed in triplicate using a SYBR Premix Ex TaqTM (Vazyme, Nanjing) in ABI 7500 HT Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA). The qPCR conditions were as follows: 95°C for 10 min and then 40 cycles of 95°C for 30 s, 64°C for 34 s, and 72°C for 30 s. The 2^−ΔΔCt formula method was used to analyze relative mRNA expression calibrated with β-actin as the reference gene. Primers were listed in Table 2.Table 2

Sequences of the primers for PCR.

Gene name	Accession no.	Primer sequence (5′-3′)	Product size (bp)
CCND1	NM_205,381.1	F: CCTCAAGAAAAGCCGGTTGC	86
		R: CTGCGGTCAGAGGAATCGTT
CDK2	NM_0,011,99857.1	F: TCCGTATCTTCCGCACGTTG	183
		R: GCTTGTTGGGATCGTAGTGC
Caspase-3	NM_204,725.1	F: CAGCTGAAGGCTCCTGGTTT	98
		R: GCCACTCTGCGATTTACACG
VEGFA	NM_0,011,10355.1	F: GTCGTACATATTCAGGCCATC	197
		R: GATTCTTTGGTCTGCAGTCAC
NOS3	JQ434761.1	F: GAACCCCCAAGACCTACGTGC	180
		R: CCTGCCCCATGGTCATTCCTC
β-actin	NM_205,518	F: ACACCCACACCCCTGTGATGAA	136
		R: TGCTGCTGACACCTTCACCATTC

Total RNA was extracted using TRIzol^® reagent (Sigma-Aldrich; Merck KGaA). cDNA was synthesized from 2 µg total RNA using ReverTra Ace^® qPCR Reverse Transcriptase kit (cat. no. FSQ-101; Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer's protocol. qPCR was performed using the SYBR-Green Master mix (cat. no. QPK-212; Toyobo Co., Ltd.) in an ABI 7500HT Real-Time PCR machine (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions for PCR were 95°C for 60 sec, followed by 40 cycles at 95°C for 15 sec, 61°C for 15 sec and 72°C for 45 sec. The relative expression level was determined by the 2^−∆∆Cq method and normalized against GAPDH (15 (link)). The primer sequences used were as follows: Forward, 5′-CGAATCATTGAAGATCCGAGTG-3′ and reverse, 5′-ATGGCTTAGAGCCCAATTATGT-3′ for cathepsin L; forward, 5-TAT TGC CTG ATT CTG TGG ACT G-3 and reverse, 5-TGA TGT ACTG GAA AGC CGT TGT-3 for cathepsin S; forward, 5′-GCAGATCGTAGCTGGGGTGAACT-3′ and reverse, 5′-AAGCAAGAAGGAAGGAGGGAGGG-3′ for cystatin C; and forward, 5′-TGCACCACCAACTGCTTAGC-3′ and reverse, 5′-GGCATGGACTGTGGTCATGAG-3′ for GAPDH.