TRIzol (Vazyme, Nanjing, China) was used to extract the total RNA of ovine adipocytes and the eight tissues collected. The cDNAs were synthesized using a HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). U6 [23 (link)] and TBP [24 (link)] were chosen as internal references to normalize the expression of miRNAs and mRNAs, respectively. The RT-qPCR was performed in triplicate using the 2× ChamQ SYBR qPCR Master system (Vazyme, Nanjing, China) on an Applied Biosystems QuantStudio 6 Flex Real-time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The information of PCR primers is listed in Table S1 . The relative expression level of the RNA was calculated using a 2−ΔΔCt method.
Applied biosystems quantstudio 6 flex real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Singapore
The Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR System is a laboratory instrument designed for quantitative real-time PCR (polymerase chain reaction) analysis. The system's core function is to precisely quantify and analyze nucleic acid samples in a real-time manner.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (14 mentions)
Trizol, by Thermo Fisher Scientific (13 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (12 mentions)
Rneasy mini kit, by Qiagen (8 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (8 mentions)
Hiscript 3 1st strand cdna synthesis kit, by Vazyme (6 mentions)
Talent qpcr premix sybr green, by Tiangen Biotech (6 mentions)
Phasemaker tubes complete system, by Thermo Fisher Scientific (6 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Taqman probe, by Thermo Fisher Scientific (6 mentions)
Mastercycler thermocycler, by Eppendorf (5 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
Superreal premix plus sybr green kit, by Tiangen Biotech (5 mentions)
Cdna synthesis kit, by Takara Bio (4 mentions)
Tb green premix ex taq kit, by Takara Bio (4 mentions)
Chamq sybr qpcr master system, by Vazyme (3 mentions)
Trizol, by Vazyme (3 mentions)
Sybr green qpcr master mix, by Vazyme (3 mentions)
Nanodrop 8000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Transscript one step gdna removal and cdna synthesis supermix, by Transgene (3 mentions)
30 protocols using applied biosystems quantstudio 6 flex real time pcr system
1
Ovine Adipocyte RNA Extraction and qPCR
2
Quantitative Analysis of Gene Expression in HUVECs
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Total RNA from HUVECs were extracted using an RNeasy Mini Kit according to the manufacturer's instructions. cDNA was synthesized using a Maxima First Strand cDNA Synthesis kit. 2 μg RNA was mixed with 2 μl Maxima Enzyme Mix, and the 4 μl reaction mix (5x) contains the remaining reaction components: reaction buffer, dNTPs, oligo(dT)18, and random hexamer primers, in total a 20 μl reaction volume. cDNA synthesis reaction was performed at 25°C for 10 min, 50°C for 30 min, and 85°C for 5 min. Real-time PCR was performed in triplicate in the Applied Biosystems™ QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific, Singapore). A final 20 μl volume included 2 μl cDNA; 10 μl Maxima SYBR Green/ROX qPCR Master Mix (2x) containing Maxima Hot Start Taq DNA Polymerase, SYBR Green I, ROX passive reference dye, and dNTPs (also dUTP) in an optimized PCR buffer; and 1 μM each of the respective forward and reverse primers. Reactions were performed at 50°C for 2 min and 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s, and annealing and extension at 60°C for 30 s and 72°C for 30 s. The amount of the target gene was normalized to GAPDH and was calculated by the 2−ΔΔCT method. Results were expressed as the fold change to the control group. The primers used for real-time PCR are listed in Table 1 .
3
RNA Isolation, cDNA Synthesis, and XBP1 Splicing Analysis
4
Quantifying Caprine Circular RNA Expression
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Total RNA from nine caprine tissues and SMSCs was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The concentration and purity of the RNA was checked using a NanoDrop 8000 spectrophotometer (NanoDrop Technologies, Wilmington, NC, USA). The cDNA was synthesized with a HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China).
The RT-qPCR analysis was performed in triplicate with the 2 × SYBR Green qPCR Master Mix (Vazyme, Nanjing, China) on an Applied Biosystems QuantStudio 6 Flex Real-time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). GAPDH was used as an internal control to normalize the expression of circ_003628 and its parent genes [19 (link)]. The 2−ΔΔct method was used to calculate their relative expression levels. The information of RT-qPCR primers is listed inTable 1 .
The RT-qPCR analysis was performed in triplicate with the 2 × SYBR Green qPCR Master Mix (Vazyme, Nanjing, China) on an Applied Biosystems QuantStudio 6 Flex Real-time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). GAPDH was used as an internal control to normalize the expression of circ_003628 and its parent genes [19 (link)]. The 2−ΔΔct method was used to calculate their relative expression levels. The information of RT-qPCR primers is listed in
5
Curcumenol modulation of TNFα-induced gene expression
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NP cell line and primary NP cells were stimulated with TNFα (10 ng/ml) with different concentrations of curcumenol (0, 6.25, 12.5, 25, and 50 μM, dissolved in DMSO; bought from Selleck Chemicals, Houston, TX, United States; with the following characteristics: high performance liquid chromatography, purity = 99.89%; nuclear magnetic resonance, consistent structure) for 24 h at 37°C with 5% CO2. Then, total RNA was isolated from the cells using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, United States) as per the manufacturer’s protocol. First strand complementary DNAs (cDNAs) were reverse transcribed from the extracted RNAs using the cDNA Synthesis Kit (Takara Bio, Otsu, Japan). Real-time qPCR was conducted using the TB Green Premix Ex Taq Kit (Takara Bio) on an Applied Biosystems QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific) per the following conditions: denaturation at 95°C for 30 s; 40 cycles of 95°C for 3 s and 60°C for 34 s; and then 95°C for 15 s, 60°C for 60 s, and finally, 95°C for 15 s. Specific primer pairs were designed using NCBI BLAST and sequences provided in Table 1 . The gene expression of β-actin was used as an internal control. Target gene expression levels were determined using the 2−ΔΔCT method.
6
Real-Time qPCR Gene Expression Analysis
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RNA extraction was conducted using TRIzol reagent and the Phasemaker Tubes Complete System (Thermo Fisher Scientific), as previously described (Chen et al., 2020 ). Thereafter, TransScript One‐Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech) was used to synthesize the cDNA for each RNA sample. The qPCR assay was performed using Talent qPCR PreMix (SYBR Green) (Tiangen Biotech) and the Applied Biosystems QuantStudio 6 Flex Real‐Time PCR System (Thermo Fisher Scientific), according to the manufacturers’ protocol. The internal reference gene was gapA and the relative level of gene expression was calculated using the ΔΔCt method, according to the MIQE guidelines (Taylor et al., 2010 (link)). The primers for the SYBR Green qPCR were designed using Beacon designer software (http://www.premierbiosoft.com/molecular_beacons/ ) and are listed in Table S2 .
7
Quantitative RNA Expression Analysis
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Total RNA was extracted with TRIzol (Thermo fisher scientific) following the manufacturer’s instructions and reverse transcribed with Hifair® II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (Yeason, Shanghai, China). cDNA was quantified using TB Green® Premix Ex Taq™ (Tli RNase H Plus) (Takara) with an Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR System (Thermo fisher scientific) following the manufacturer’s instructions.
8
Quantitative Gene Expression Analysis
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Total RNA from isolated glomeruli, tubules, PTCs, or TECs was extracted using the RNeasy kit (QIAGEN) and then reverse-transcribed using an RT-PCR Kit (Superscript III; Invitrogen). Gene expression was evaluated by quantitative real-time PCR with SYBR Green PCR Master Mix (Qiagen) in an Applied Biosystems QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific) using mouse 18s rRNA or β-actin as an internal control. Quantitative PCR was conducted in triplicates for each sample. Primer sequences are provided in SI Appendix, Table 1 .
9
Quantitative RT-PCR Analysis of c-di-GMP Genes
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Bacterial cells grown in LB medium (OD600 = 0.6) were harvested, and total RNAs were isolated using TRIzol reagent and the Phasemaker Tubes complete system (Invitrogen, Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. RNA quality and integrity assessment were performed using the NanoDrop 2000c system (Thermo Fisher Scientific, USA) and agarose gel electrophoresis.
For cDNA synthesis, 1 μg total RNA of each sample was used as the template to synthesize cDNA using the FastQuant RT kit (with genomic DNase) (Tiangen Biotech, China). Talent qPCR PreMix (SYBR green) (Tiangen Biotech, China) was used for qRT-PCR to quantify the transcript levels of the c-di-GMP turnover gene, with gapA or recA used as the internal reference gene according to MIQE guidelines (59 (link), 60 (link)). Primers for SYBR green qRT-PCR were designed using the Beacon designer (Premier Biosoft) and are listed inTable S2 . Assays were performed in quadruplicate in 10-μl reaction mixtures using the Applied Biosystems QuantStudio 6 Flex real-time PCR system (Thermo Fisher Scientific, USA). PCR was performed according to the manufacturer’s instructions. The gene expression level was analyzed and calculated using ΔΔCT methods as previously described (61 (link)). Data represent the means from three independent biological repeats, with error bars indicating the standard deviations.
For cDNA synthesis, 1 μg total RNA of each sample was used as the template to synthesize cDNA using the FastQuant RT kit (with genomic DNase) (Tiangen Biotech, China). Talent qPCR PreMix (SYBR green) (Tiangen Biotech, China) was used for qRT-PCR to quantify the transcript levels of the c-di-GMP turnover gene, with gapA or recA used as the internal reference gene according to MIQE guidelines (59 (link), 60 (link)). Primers for SYBR green qRT-PCR were designed using the Beacon designer (Premier Biosoft) and are listed in
10
Quantitative Real-Time PCR Gene Expression Analysis
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A total of 2 μg total RNA in a 20 μl reaction was converted to cDNA with SuperScript III Reverse Transcriptase (Invitrogen, United States) according to the manufacturer’s instructions on an Eppendorf Mastercycler thermocycler (Eppendorf AG, Germany) with the following conditions: 25°C for 5 min, 50°C for 60 min, and 70°C for 15 min, followed by a hold at 4°C until use in a Quantitative real-time PCR (qPCR) reaction. A total of 60 μl of deionized water was added to 20 μl cDNA, and 1 μl of diluted cDNA mixture was used as the input for the qPCR reaction. The qPCR reactions were performed with a SuperReal PreMix Plus SYBR Green Kit (TIANGEN Biotech, Beijing, China), following the manufacturer’s instructions in a 20 μl volume. The qPCR was performed on an Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, United States) with the following cycling conditions: 95°C for 15 min, followed by 40 cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 32 s. The melt curve conditions were 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. All samples had only one melt temperature peak. The Log2Foldchange was calculated using the 2–ΔΔCT method with 26S as a reference gene. The CT values represent the average of the three technical replicates. The sequences of primers used for qRT-PCR are listed in Supplementary Table 4 .
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