Total RNA was purified from periodontal tissue with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was generated using PrimeScript RT Master Mix (Toyobo Co, Ltd, Osaka, Japan). The cells isolated by FACS were lysed, and reverse transcription was performed using a SYBR™ Green Fast Advanced Cells-to-CT™ Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Gene expression levels were measured by RT-qPCR using a BioRad CFX96TM Detection System (Roche, Sweden) and SYBR PCR Master Mix (Roche, Indianapolis, IN, USA). The primers used in this experiment are shown in Supplementary Table 2 .
Cfx96 detection system
Manufactured by Roche
Sourced in Sweden
The CFX96 detection system is a real-time PCR instrument manufactured by Roche. It is designed for the quantitative detection and analysis of nucleic acid sequences. The system utilizes fluorescence-based detection technology to monitor the amplification of target DNA or RNA sequences in real-time. The CFX96 detection system is a versatile laboratory tool suitable for a range of applications in molecular biology, genetics, and diagnostic research.
Automatically generated - may contain errors
Lab products found in correlation
Sybr pcr master mix, by Roche (4 mentions)
Primescript rt master mix, by Toyobo (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Nucleozol reagent, by Macherey-Nagel (2 mentions)
Mirna isolation kit, by Qiagen (2 mentions)
Sybr green fast advanced cells to ct kit, by Thermo Fisher Scientific (1 mentions)
Cfx96 detection system, by Bio-Rad (1 mentions)
Sybr pcr master mix, by Bio-Rad (1 mentions)
Primescript rt kit, by Takara Bio (1 mentions)
Legendplex bead based immunoassay, by BioLegend (1 mentions)
Nucleozol reagent, by Gene Tech (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Hieff qpcr sybr green master mix, by Yeasen (1 mentions)
6 protocols using cfx96 detection system
1
RNA Isolation and RT-qPCR Analysis of Periodontal Tissue
2
Quantifying Gene Expression by qRT-PCR
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Total RNA was extracted and purified by NucleoZol reagent (Macherey–Nagel), and cDNA was obtained by a cDNA library kit (Takara). qRT-PCR was conducted on a Bio-Rad CFX96TM detection system (Roche) with SYBR PCR Master Mix (Roche). β-Actin was used for standardization. The primers used are listed in Additional file 1 : Table S2.
3
Quantifying Gene and miRNA Expression
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Total RNA was extracted from periodontal tissue and cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was generated with PrimeScript RT Master Mix (Toyobo Co, Ltd, Osaka, Japan). The expression level of genes was measured by qPCR in a Bio-Rad CFX96™ Detection System (Roche, Sweden) with SYBR PCR Master Mix (Roche, Indianapolis, IN, USA). Small RNA was extracted from cells with an miRNA isolation kit (Qiagen, Hilden, Germany), and cDNA was generated with an miRNA reverse transcription kit (Shenggong, Shanghai, China). The expression level of miRNAs was measured by qPCR in a Bio-Rad CFX96™ Detection System with SYBR PCR Master Mix. U6 was used as the internal reference. The primers used are shown in Supplementary Table S1 .
4
Testicular Tissue RNA and Protein Analysis
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Total RNA was purified from testicular tissue samples using Nucleozol reagent (Macherey-Nagel, 740404.200) according to the manufacturer’s protocol. Total RNA was reverse transcribed into cDNA using the Prime Script RT kit (TaKaRa, E237). Quantitative PCR (qPCR) was performed using SYBR mix (ChamQ, Q311-02) according to the manufacturer’s instructions. Signals were detected using a Bio–Rad CFX96 detection system (Roche). The primers that were used for this study are outlined in Table 1
Testicular tissues were lysed in ice-cold RIPA lysis buffer (Elabscience®, E-BC-R327) and the supernatants were collected. The supernatants protein concentrations were normalized using the BCA assay. Protein levels were analyzed using a LEGEND plex bead-based immunoassay (BioLegend, 740134) according to the manufacturer’s instructions. The mean fluorescence intensity of the cytokines was analyzed using LEGEND plex8.0 software.
Testicular tissues were lysed in ice-cold RIPA lysis buffer (Elabscience®, E-BC-R327) and the supernatants were collected. The supernatants protein concentrations were normalized using the BCA assay. Protein levels were analyzed using a LEGEND plex bead-based immunoassay (BioLegend, 740134) according to the manufacturer’s instructions. The mean fluorescence intensity of the cytokines was analyzed using LEGEND plex8.0 software.
5
RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted from the colons, the gingivae and CD4+ T cells with NucleoZOL reagent (Gene Company Limited) and was then reverse-transcribed into cDNA using PrimeScript RT Master Mix (TaKaRa, Ltd, Osaka, Japan). Real-time polymerase chain reaction (RT-qPCR) was performed to measure gene expression levels in a Bio-Rad CFX96™ detection system (Roche, Sweden) with Hieff qPCR SYBR Green Master Mix (Yeasen, Shanghai, China). Small RNA was isolated with a miRNA isolation kit (Qiagen, Hilden, Germany), and cDNA was prepared with a miRNA reverse transcription kit (Shenggong, Shanghai, China). RT-qPCR was performed to measure the expression level of genes in a Bio-Rad CFX96™ detection system (Roche) with Hieff qPCR SYBR Green Master Mix (Yeasen). U6 was applied as the internal reference. The primers used in the process are shown in Supplementary Table S1 .
6
Analysis of ER Stress Markers in HGECs
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HGECs were suspended in 1 mL of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and stored at - 80°C.
Total RNA was puri ed using TRIzol reagent following the manufacturer's instructions and quanti ed with a Nanodrop 2000. cDNA was synthesized from 500 ng of total RNA using Prime Script RT Master Mix (Toyobo Co, Ltd, Osaka, Japan). Real-time PCR was performed in a 96-well optical reaction plate using SYBR PCR Master Mix (Roche, Indianapolis, IN, USA). mRNA expression was assayed on a Bio-Rad CFX96 detection system (Roche, Sweden). Details of the RT-qPCR primers used in this experiment are shown in Table 1. Relative SERPINH1, GRP78, IRE1, TRAF2, NLRP3, IL-1β, XBP1-s and XBP1-u expression was quanti ed with the 2-ΔΔCt method using GAPDH expression as an endogenous control.
Total RNA was puri ed using TRIzol reagent following the manufacturer's instructions and quanti ed with a Nanodrop 2000. cDNA was synthesized from 500 ng of total RNA using Prime Script RT Master Mix (Toyobo Co, Ltd, Osaka, Japan). Real-time PCR was performed in a 96-well optical reaction plate using SYBR PCR Master Mix (Roche, Indianapolis, IN, USA). mRNA expression was assayed on a Bio-Rad CFX96 detection system (Roche, Sweden). Details of the RT-qPCR primers used in this experiment are shown in Table 1. Relative SERPINH1, GRP78, IRE1, TRAF2, NLRP3, IL-1β, XBP1-s and XBP1-u expression was quanti ed with the 2-ΔΔCt method using GAPDH expression as an endogenous control.
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