Total RNA was extracted from HRECs and retinas using a universal RNA Purification Kit (B004, EZ Bioscience, USA) following the manufacturer’s protocol. The concentration and quality of RNA were examined using Nanodrop 2000 (Thermo Fisher Scientific). RNA samples were reverse-transcribed to complementary DNA (cDNA) using a Colour Reverse Transcription Kit (A0010CGQ, EZBioscience). Quantitative polymerase chain reaction (qPCR) was performed using SYBR Green Master Mix (A0012, EZBioscience). GAPDH was used as an internal reference for each reaction. The relative expression was calculated using the following equation: Relative gene expression = 2[△Ct(control)–△Ct(target)]. The sequences of the primers are listed in Additional file 1 : Table S1.
Sybr green master mix
Manufactured by EZBioscience
Sourced in United States
SYBR Green Master Mix is a ready-to-use solution for real-time quantitative PCR (qPCR) and reverse transcription-qPCR (RT-qPCR) applications. It contains SYBR Green I dye, hot-start DNA polymerase, and necessary reagents for efficient amplification and detection of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480, by Roche (3 mentions)
Rna purification kit, by EZBioscience (2 mentions)
Reverse transcription kit, by EZBioscience (2 mentions)
Rna extraction column kit, by EZBioscience (2 mentions)
Cdna synthesis kit, by EZBioscience (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Universal rna purification kit, by EZBioscience (1 mentions)
Colour reverse transcription kit, by EZBioscience (1 mentions)
Quantstudio 7 flex system, by Thermo Fisher Scientific (1 mentions)
Quantstudio 7 flex, by Thermo Fisher Scientific (1 mentions)
Ezscript reverse transcription mix 2 kit, by EZBioscience (1 mentions)
Glucose assay kit, by Beyotime (1 mentions)
Oe cloud, by OE Biotech (1 mentions)
Bacterial rna extraction kit, by Vazyme (1 mentions)
Nad nadh assay kit, by Beyotime (1 mentions)
Pyruvate assay kit, by Solarbio (1 mentions)
Atp assay kit, by Beyotime (1 mentions)
8 protocols using sybr green master mix
1
Quantification of Gene Expression in HRECs
2
RNA Extraction and qRT-PCR Analysis
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The total RNA was extracted using the RNA Purification Kit (EZBioscience, CN) and was reverse transcribed into cDNA. The PCR reactions were performed using the QuantStudioTM 7 Flex System (Thermo Fisher Scientific, US) and SYBR Green Master Mix (EZBioscience, CN). The relative expression of genes was calculated using 2−ΔΔCT. GAPDH was used as a reference gene. All primers information was shown in Table S2 .
3
Real-Time Quantitative PCR Analysis of Chondrogenic Differentiation
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After 7 and 14 days of chondrogenic differentiation, total RNA was isolated and complementary DNA (cDNA) was prepared using the RNA purification kit and 4 × EZscript Reverse Transcription Mix II kit (EZBioscience, Roseville, MN, USA) according to the manufacturer’s instructions. Real-time quantitative PCR was performed using the SYBR Green Master Mix (EZBioscience, Roseville, MN, USA) in a real-time PCR System (QuantStudio™ 7 Flex, Thermo Fisher Scientific, Waltham, MA, USA). The reaction was performed with a total volume of 10 μL in each reaction well under the following conditions: 95 °C for 5 min to activate, followed by 40 cycles of 10 s at 95 °C and 60 s at 60 °C. The quantitative gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the relative expression was determined using the ΔΔCt method. Each sample has three technical replicates. Primer sequences are listed in Table 2 .
4
Transcriptomic Analysis of S. aureus Biofilm
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S. aureus biofilms were collected after different treatments and immediately stored in liquid nitrogen. The samples were detected for transcriptomic analysis with the mass spectrometry platform of OE Biotech (Shanghai, China). The data obtained were further analyzed using OE Cloud (OE Biotech) software.
qPCR was used to validate the expression levels of genes that were significantly regulated in the transcriptomics analysis results. Briefly, total S. aureus RNA was extracted from biofilms using a bacterial RNA extraction kit (Vazyme, China). cDNA was synthesized using a reverse transcription kit (EZBioscience, USA) after the determination of RNA concentration. cDNA obtained was subsequently amplified using SYBR Green Master Mix (EZBioscience, USA) and a LightCycler 480 (Roche, USA). The 16s RNA was selected as the housekeeping gene and the primers used were listed inTable S1 .
Finally, the glucose metabolism of S. aureus in biofilms was measured using the Glucose Assay Kit (Beyotime, China), Pyruvate Assay Kit (Solarbio, China), ATP Assay Kit (Beyotime, China), and NAD+/NADH Assay Kit (Beyotime, China), respectively.
qPCR was used to validate the expression levels of genes that were significantly regulated in the transcriptomics analysis results. Briefly, total S. aureus RNA was extracted from biofilms using a bacterial RNA extraction kit (Vazyme, China). cDNA was synthesized using a reverse transcription kit (EZBioscience, USA) after the determination of RNA concentration. cDNA obtained was subsequently amplified using SYBR Green Master Mix (EZBioscience, USA) and a LightCycler 480 (Roche, USA). The 16s RNA was selected as the housekeeping gene and the primers used were listed in
Finally, the glucose metabolism of S. aureus in biofilms was measured using the Glucose Assay Kit (Beyotime, China), Pyruvate Assay Kit (Solarbio, China), ATP Assay Kit (Beyotime, China), and NAD+/NADH Assay Kit (Beyotime, China), respectively.
5
Gene Expression Analysis by qRT-PCR
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Total RNA of the cultured cells was extracted using an RNA Purification Kit (EZBioscience, Roseville, MN, USA). After measuring the concentration of RNA, Complementary DNA was synthesized using a Reverse Transcription Kit (EZBioscience) and used as template to perform qRT-PCR using SYBR Green Master Mix (EZBioscience). Primers were obtained from Sangon Biotech (Shanghai, China) (Table 1 ). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as internal control.
6
Quantitative gene expression analysis
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Total RNA was extracted and cDNA was acquired according to the manufacturer's instructions (Thermo Scientific). qPCR was performed using SYBR Green Master Mix (EZBioscience) according to the instructions for the PCR amplifier (Bio‐Rad).
7
RNA Extraction and qRT-PCR Analysis of BMDMs
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After 24 h of incubation, total RNA was extracted from BMDMs in the four groups using an RNA extraction column kit (EZBioscience, USA) according to the manufacturer’s instructions. Subsequently, complementary DNA was synthesized from 1 μg of total RNA using a cDNA synthesis kit (EZBioscience, USA). Quantitative RT-PCR was performed using a SYBR Green Master mix (EZBioscience, USA) on LightCycler 480 (Roche, USA). The primers used are shown in Additional file 1 : Table S1.
8
RNA Extraction and qRT-PCR Analysis
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After 24 h of incubation, the total RNA was extracted using an RNA extraction column kit (EZBioscience, USA) according to the manufacturer's instructions. Subsequently, complementary DNA was synthesized from 1 μg of total RNA using a cDNA synthesis kit (EZBioscience, USA). Quantitative RT-PCR was performed using a SYBR Green Master mix (EZBioscience, USA) on LightCycler 480 (Roche, USA). The primers used are shown in Table S1.
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