Rna extraction kit

Total RNA was extracted from cells using RNA extraction kit (Accurate Biology, AG21024) according to the manufacturer's protocol. Complementary DNA (cDNA) was prepared using the cDNA Synthesis SuperMix for qPCR (YEASEN, 11141ES60). Real‐time quantitative PCR was performed using SYBR GreenMaster Mix (YEASEN, 11184ES50). The primer sequence for the genes used in this study were as follows: GAPDH (forward, 5′‐TGTCGTGGAGTCTACTGGTG‐3′; reverse, 5′‐GCATTGCTGACAATCTTGAG‐3′), p16^INK4a (forward, 5′‐GAAAGAGTTCGGGGCGTTG‐3′; reverse, 5′‐GAGAGCCATCTGGAGCAGCAT‐3′), p21 (forward, 5′‐GTGGGTGTCAAAGCACTTAG‐3′; reverse, 5′‐ACAGTCCAGACCAGGATGTTA‐3′), p53 (forward, 5′‐ATCGCCTTCGACATCATCGC‐3′; reverse, 5′‐CCCCATGCGTACTCCATGAG‐3′), FOXO1 (forward, 5′‐ CTACGAGTGGATGGTGAAGAGC‐3′; reverse, 5′‐CCAGTTCCTTCATTCTGCACTCG‐3′), and REDD1 (forward, 5′‐ CAAGGCAAGAGCTGCCATAG‐3′; reverse, 5′‐CCGGTACTTAGCGTCAGGG‐3′). The relative expression of each target gene was calculated using 2^−ΔΔCT method with GAPDH for normalization.

RNA was extracted from chondrocytes using an RNA extraction kit (Accurate Biology, Hunan, China), and the RNA concentration was detected by UV spectrophotometer and stored at − 80 ℃. The expression of TUG1, DUSP1, miR-144-3p, MMp13, and collagen II was detected by SYBR Green QPCR master mix (Beyotime Biotechnology, Shanghai, China). Using β-actin as the internal reference control, the average CT value (amplification power curve inflection point) was taken and semi-quantitative analysis was performed by 2^−△△Ct to calculate the relative expression of target genes. The primer sequences are shown in Table 2.Table 2

The primer sequences

Gene	Primer sequence
Collagen II	Forward: GAGCCCTGCCGGATCTGT Reverse: GAGGCAGTCTTTCACGTCTTC
MMP13	Forward: TCCTGATGTGGGTGAATACAATG Reverse: GCCATCGTGAAGTCTGGTAAAAT
TUG1	Forward: CACTCATCCTGTGCCTCCTG Reverse: TGATGGCTGAATGCCTCCTG
miR144-3P	RT GTCGTATCCAGTGCAGGGTCCGAGG TATTCGCACTGGATACGACAGTACA Forward: GCGCGCTACAGTATAGATGA Reverse: GTGCAGGGTCCGAGGT
Dusp1	Forward: GCATGGTCATGGAAGTGGGCAC Reverse: GTCAGCAGCTGGGAGAGGTCG
β-Actin	Forward: CTCCATCCTGGCCTCGCTGT Reverse: GCTGTCACCTTCACCGTTCC
U6	RT AACGCTTCACGAATTTGCGT Forward: CTCGCTTCGGCAGCACA Reverse: AACGCTTCACGAATTTGCGT

Total RNA was extracted from clinical specimens using an RNA extraction kit (Accurate Biology, AG21017). Evo M-MLV RT Premix (Accurate Biology, AG11706) and SYBR Premix Ex TaqTM (Accurate Biology, AG11702) were used to prepare samples for RT-qPCR, which was conducted using a Rotor-Gene Q instrument (Qiagen, Germany). We calculated the relative expression levels of target genes based on the 2^-ΔΔCT method. The primers used were as follows: human β-actin Forward: 5’– GGCACCCAGCACAATGAA–3’; human β-actin Reverse: 5’– TAGAAGCATTTGCGGTGG–3’; human GPX4 Forward: 5’– ATCGACGGGCACATGGTTAA–3’; human GPX4 Reverse: 5’– CAGGATCCGCAAACCACACT–3’.

The total RNA was extracted from cells using an RNA Extraction Kit (Accurate Biotechnology, Danyang, China). Total RNA was reverse-transcribed to cDNA using a one-step RT-PCR kit (Accurate Biotechnology, China). RT-qPCR was performed using SYBR Green (Accurate Biotechnology, China) in an RT-PCR machine (Agilent Technologies, Santa Clara, CA, USA). Values were normalized to GAPDH mRNA levels. The gene primer sequences used for qRT-PCR are listed in Table 1.

Retinal tissues were collected from D0 control mice and D14 EAU mice, and total RNA was isolated using an RNA extraction kit (Accurate Biology, AG21023). For RT‐qPCR, total RNA was reverse‐transcribed using RT Master Mix for qPCR (MCE, HY‐K0510). cDNA was quantified using primers specific for mice in an ABI 7500 real‐time PCR system (Applied Biosystems). PCR amplification was performed in a volume of 20 µL using the SYBR Green qPCR Master Mix (MCE, HY‐K0501). The specific transcripts were confirmed by assessment of melting curve profiles at the end of each PCR round. The results were analyzed based on group assignments. β‐Actin was used as the internal control, and the results were calculated using the ^ΔΔCt method.

The mRNA levels of runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), collagen type I (COL-1), and bone morphogenetic protein 2 (BMP2) were assessed by qRT-PCR. The cells were first washed three times with cold PBS, and total RNA was isolated using the RNA Extraction Kit (Accurate Biology, AG21017). cDNA was synthesized using the Total Transcriptome CDNA Synthesis Kit (Accurate Biology, AG11707). cDNAs were normalized to GAPDH, respectively. Primers were designed with Primer Premier 5.0 software and listed in Table 1. A comparison of the 2^−ΔΔCt method was used to quantify the relative expression levels of mRNAs.