Through lysis and centrifugation, total RNAs of fresh cells or tissues (frozen at −80 °C) were extracted using a total RNA small extraction kit (B618583-0100, Sangon, China). Total RNA was treated with RNase-free DNaseI. RNase-free water (20 µL; Am9932, Thermo Fisher Scientific, Waltham, MA, USA) was used to dissolve the total RNA, and an ultra-micro spectrophotometer (Nanodrop One, Thermo Fisher Scientific, Waltham, MA, USA) was used to determine its concentration and purity. Samples with ratios of 260/280 nm values between 1.9 and 2.1 and 260/230 nm values greater than 2.0, were used for subsequent experiments. HiFiScript cDNA Synthesis Kit (CW2569, CWBIO, Jiangsu, China) and SYBR Green qPCR Kit (11201ES08, Yeasen, Shanghai, China) were used for reverse transcription and stored at −20 °C pending analysis. Subsequently, quantitative real-time PCR (qRT-PCR) was performed using a LightCycler96 qRT-PCR instrument (Roche, Basel, Switzerland). The cycling conditions were determined according to the manufacturer’s instructions. β-actin was used as an endogenous control. All data were processed by relative quantitative method (2−△△Ct). The primer sequences are listed in Table 1 . Genomic DNA contamination was not detected in the qRT-PCR products.
Sybr green qpcr kit
Manufactured by Yeasen
Sourced in China
The SYBR Green qPCR Kit is a reagent system designed for quantitative real-time PCR (qPCR) analysis. It contains SYBR Green I dye, which binds to double-stranded DNA and emits a fluorescent signal that can be detected and measured during the PCR process. The kit provides the necessary components for performing qPCR reactions, including DNA polymerase, dNTPs, and reaction buffer.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Am9932, by Thermo Fisher Scientific (1 mentions)
Lightcycler 96 qrt pcr instrument, by Roche (1 mentions)
Nanodrop one, by Thermo Fisher Scientific (1 mentions)
Hifiscript cdna synthesis kit, by CWBIO (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Rna extraction kit, by Yeasen (1 mentions)
Reverse transcription kit, by Yeasen (1 mentions)
Reverse transcriptase kit, by Yeasen (1 mentions)
7 protocols using sybr green qpcr kit
1
RNA Extraction and qRT-PCR Analysis
2
Validating Lipid Metabolism Genes
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To validate RNA-seq results, 12 lipid metabolism genes, including 6 genes from the N. bombycis and 6 genes from the silkworms, were selected for relative expression analyses using RT-qPCR assays. The RT-qPCR primers are shown in Supplementary Table 1 . For RT-qPCR analysis, qPCR reactions (95 °C for 5 min, followed by 40 cycles of 95 °C for 20 s, 60 °C for 30 s, and 72 °C for 20 s) were performed using SYBR Green qPCR kit (Yeasen, Shanghai, China). Tubulin and β-actin were used as the endogenous control of N. bombycis and B. mori, respectively. All qPCR experiments were conducted in triplicate.
3
Quantitative Real-Time PCR Analysis
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Total RNA from the clinical specimens was extracted using TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s instructions. Total RNA from the cells was extracted using an RNA extraction kit (19221ES50, Yeasen, China). The quantity and quality of RNAs were detected by A260/A280 with a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA, USA). Two micrograms of total RNA was used to synthesize cDNA (11141ES60, Yeasen, China). RT-qPCR was performed using a SYBR Green qPCR kit (11199ES08, Yeasen, China). Relative mRNA expression was normalized to that of the internal GAPDH control. The primer sequences are listed in Supplementary Table S1
4
Quantitative Analysis of mRNA Expression
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Total RNA was extracted using TRIzol reagent (Invitrogen), followed by cDNA synthesis (1,000–2,000 ng total RNA samples) using a reverse transcription kit (Yeasen, China). Quantitative real-time PCR was performed using a SYBR Green qPCR kit (Yeasen, China), and the primers were designed and synthesized by Tsingke Biotech Com (Shanghai, China). Relative mRNA expression was normalized to GAPDH mRNA and then calculated using the 2−△△CT method. The primer sets used are shown in Supplementary Table S1.
5
Quantifying Inflammatory Cytokine Expression in THP-1 Cells
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TRIzol reagent (Cat. 15596026, Invitrogen) was used to extract total RNA. Reverse-transcribe total RNA (2 g) into complementary DNA (cDNA) for mRNA assays by using a first-strand cDNA synthesis kit (Cat. 11119ES60, Yeasen), Subsequently, THP-1 cells induced with different concentrations of MSU (0 μg/ml, 50 μg/ml and 100 μg/ml) 24 (link), using the SYBR Green qPCR Kit (Cat. 11203ES03) to detect the concentrations of IL1A and three pro-inflammatory cytokines (IL-8, IL-1β, TNFα), qRT-PCR analysis was carried out on a TL998-IV Real-Time PCR System. As an internal control, GAPDH was used. The 2-ΔΔCt method was used to examine the findings of qRT-PCR. The primers sequences were as follows: IL1A-forward, 5'-ATCATGTAAGCTATGGCCCACT-3′, IL1A-reverse, 5'-CTTCCCGTTGGTTGCTACTAC-3′; IL-1β-forward, 5'-TCTGGTAATCCACTCAAATAGGGA, IL-1β-reverse, 5'-ACCTTGTGATGTAGTGTTGTGGT-3′; IL-8-forward, 5'-TGAGCAGATCTTGCATGTAGC-3′, IL-8-reverse, 5′-GCTGCTGATCAAGAAGATTCACC-3′; TNFα-forward, 5′-GGACACTGGCCACTTACGAA-3′, TNFα-reverse, 5′-GCCAACCTTCCCAAAACCAC-3′; TLR2-forward, 5′-AATTGTGACCAAGACGGGACA-3′, TLR2-reverse, 5′-GTTGACTGGTGAGCGACGA-3′; TLR4-forward, 5'-TTGTGCAAACTTGCCGGGAGGA-3', TLR4-reverse, 5'-ACTTCTCCTTCAGCTTGGCAGC-3'; MyD88-forward, 5'-CCAACGCCAGCAAAGTTCTC-3', MyD88-reverse, 5'-AGGTCCACACAAAACCCCTG-3'; GAPDH-forward, 5′-GCACCGTCAAGGCTGAGAAC-3′, GAPDH-reverse, 5′-TGGTGAAGACGCCAGTGGA-3′.
6
Quantitative Analysis of Gene Expression
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A total of 48 h were spent cultivating raw264.7 and nucleus pulposus cells in the test media. Following the directions provided by the manufacturer, a reverse transcriptase kit (Yeasen, China) was used to extract total RNA. After determining the RNA concentration, reverse transcriptase was used to produce cDNA. After that, qRT-PCR was carried out using a SYBR Green qPCR Kit (Yeasen, China). The primer sequences of target genes (Qingke, Wuhan, China) are shown in Table 2 . The primer sequence of aggrecan aimed at the G1-B’ and IGD domains of the aggrecan gene.
7
Quantifying miRNA and mRNA Expression
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Total RNA was measured using a rapid RNA extraction kit (Epizyme, Shanghai). The purity and concentration of RNA were measured using a NanoDrop 2000 spectrophotometer. The cDNA was synthesized using a Bulge Loop™ miRNA RT primer kit (RiboBio, China). An SYBR green qPCR kit (Yeason, Guangzhou, China) was used for qRT-PCR. Expressions of miR-101-3p and EZH2 were normalized to GAPDH or U6 applying the 2-ΔΔCt method.
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