Dulbecco's modified Eagle medium (DMEM), fetal bovine serum were purchased from Corning Inc. (CA, USA). Penicillin and streptomycin were procured from Hyclone (Logan, Utah, USA). Simvastatin, oil-red O, oleic acid and dimethyl Sulphoxide DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chlorogenic acid was purchased from the Energy Chemical Company. The kit for Triglyceride (TG) was purchased from Jian Cheng Biotechnology Company (Nanjing, China). Total RNA extraction reagent Trizol was purchased from invitrogen (Carlsbad, CA, USA), PrimeScript RT reagent kit and SYBR-Green PCR kit were purchased from Transgene Biotech, Inc. (Beijing, China).
Sybr green pcr kit
Manufactured by Transgene
Sourced in China, United States
The SYBR Green PCR kit is a reagent solution designed for real-time quantitative PCR (qPCR) analysis. The kit contains SYBR Green, a fluorescent dye that binds to double-stranded DNA, enabling the detection and quantification of PCR amplification products.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Cdna reverse transcription kit, by Transgene (2 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Fastquant rt kit, by Tiangen Biotech (2 mentions)
Oleic acid, by Merck Group (1 mentions)
Simvastatin, by Merck Group (1 mentions)
Primescript rt reagent kit, by Transgene (1 mentions)
Dimethyl sulphoxide dmso, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Abi 7500 fast thermocycler, by Thermo Fisher Scientific (1 mentions)
Gdna eraser, by Takara Bio (1 mentions)
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Reverse transcription kit, by Promega (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Nanophotometer n50, by Implen (1 mentions)
Cfx96 rt pcr system, by Bio-Rad (1 mentions)
15 protocols using sybr green pcr kit
1
Statin-induced Lipid Homeostasis Regulation
2
Quantitative Real-Time PCR for miRNA Expression
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TRIzol reagent (Takara Biotechnology Co. Ltd., Dalian, China) was used to extract total RNA from cells in all groups. Afterwards, the PrimeScript RT reagent kit equipped with gDNA Eraser (Takara Biotechnology Co., Ltd.) was utilized to synthesize cDNA through reverse transcription of RNA. The ABI 7500 Fast thermocycler (Applied Biosystems, Foster City, CA, USA) was employed for PCR amplification using SYBR-Green PCR kit (TransGen Biotech Co., Ltd., Beijing, China). Following were the PCR amplification conditions: 10 min at 95°C, then 15 s at 95°C for 40 cycles, and 45 s at 60°C for annealing/extension. Shanghai Sangon (Shanghai, China) was responsible for primer design. The sequences of specific primers for all genes are as follows: for miR-200b-3p: 5′-TAATACTGCCTGGTAATGATG-3′ and 5′-CTCAACTGGTGTCGTGGA-3′; for miR-200c-3p: 5′-TAATACTGCCGGGTAATGATGG-3′ and 5′-CCTCAACTGGTGTCGTGGA-3′; for miR-429-3p: 5′-TAATACTGTCTGGTAATGCCG-3′ and 5′-CTCAACTGGTGTCGTGGA-3′, and for U6: 5′-CTCGCTTCGGCAGCACA-3′ and 5′-AACGCTTCACGAATTTGCGT-3′. Typically, gene expression was normalized relative to U6 and quantified by the 2−∆∆Ct = [(CTgene of interest-CTinternal control)sample A-(CTgene of interest-CTinternal control)sample B].
3
Cisplatin-Induced NSCLC: Tissue and RNA Analysis
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Cisplatin-induced NSCLC in patients (n=6) were collected, and cisplatin-induced NSCLC and paracarcinoma tissue were collected and saved at −80°C. The present study was approved by the Ethics Committee of The People's Hospital of Bozhou (n. PHBZ/L-2015008). All patients provided informed written consents to participate in this study. The charascteristics of the patients involved in the present study are shown in Table I . Total RNA was extracted from serum and cell using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA). Reverse transcription was carried out into cDNA using the M-MLV Reverse Transcription system (Takara Biotechnology Co., Ltd., Dalian, China). The ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) was used to perform qPCR with the SYBR-Green PCR kit (TransGen Biotech, Inc., Beijing, China). PCR primers used were as follows: miR-181: 5′-GCGGCAACATTCAACGCTGTCGGTGAGT-3′ and 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTG-3′; U6: 5′-CTCGCTTCGGCAGCACA-3′ and 5′-AACGCTTCACGAATTTGCGT-3′. PCR was performed at 95°C for 10 min, followed by 40 cycles of 95°C for 25 sec and 60°C for 30 sec. The relative quantification was calculated using the 2−ΔΔCt method.
4
RNA Extraction and RT-qPCR Analysis
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The tissues were ground, and cells from each group were collected. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). Subsequently, cDNA synthesis was performed using a reverse transcription kit (Promega). RT-qPCR was conducted using the SYBR-Green PCR kit (TransGen Biotech, Inc., Beijing, China). The data was analyzed using the 2−ΔΔCt method to determine relative gene expression levels.
5
Quantification of Metallothionein-1 Expression
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Total RNAs were extracted with TRIzol reagent (Takara, Dalian, China) according to the manufacturer’s instructions. The RNA purity and quality were validated by absorbance on a microvolume spectrometer (NanoPhotometer N50, Implen, Germany) at 260 and 280 nm. Only samples with ratios from 1.8 to 2.0 were accepted for the next reverse transcription reaction. Complementary DNAs (cDNAs) were prepared with the RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, USA). The primers were synthesized by Sangon Biotech (Shanghai, China): β-actin forward primer 5′-TCCTCTCCCAAGTCCACACAGG-3′, reverse primer 5′-GGGCACGAAGGCTCATCATTC-3′; MT-1 forward primer 5′-AGAGTGCAAATGCACCTCCTGC-3′, reverse primer 5′-CGGACATCAGGCACAGCAGCT-3′. Real-time polymerase chain reaction (RT-PCR) amplification reactions were prepared with the SYBR Green PCR kit (Transgen Biotech, China) and performed using the CFX96 RT-PCR system (Bio-Rad, USA). PCR products were verified using the melting curve analysis. The relative mRNA level of MT-1 was calculated with normalization to control the housekeeping gene β-actin and was reported using the 2−ΔΔct method.
6
Quantifying Expression Profiles in Cell Lines
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Total RNA was extracted from cultured SK-HEP1 cells, SMMC-7721 cells and HUVECs treated with piperlongumine using TRIzol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. Then, total RNA was reverse transcribed using a cDNA reverse transcription kit (TransGen, Guangzhou, China), and the obtained cDNA was amplified using a SYBR Green PCR kit (TransGen, Guangzhou, China). Quantitative real-time PCR analysis was performed to detect expression in samples. Each experiment was repeated at least three times. The primers used for qRT-PCR were obtained from RiboBio Co., Ltd. The expression of genes was analyzed using 2-ΔΔCT methodology. The specific content of the relevant primers used in qPCR can be found in the support information.
7
Quantifying mRNA and miRNA Expression
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Total RNA from the cultured foam cells and thoracic aorta tissues was isolated using TRIzol reagent (Invitrogen). Reverse transcription was carried out using the M-MLV Reverse Transcription system (Takara, Dalian, China). qPCR was performed using the SYBR-Green PCR kit as described by the manufacture (TransGen, Beijing, China). The primers used for PCR are listed in Table I . Amplification was performed by denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec. qPCR was carried out on an Applied Biosystems 7500 Fast Real-Time PCR system. The relative mRNA expression levels were normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene according to the 2−ΔΔCt method, and the miRNA expression levels were normalized to U6. All the PCR experiments were carried out in triplicate within each experiment, and the experiments were replicated at least 3 times.
8
Quantitative Analysis of RNA Expressions
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Total RNA was extracted from LAA tissues using TRIzol® reagent (Invitrogen), and the extracted RNA was reverse transcribed into cDNA using a FastQuant RT Kit (TianGen, Beijing, China). qRT-PCR was performed to detect the relative expression levels of lncRNAs, miRNAs, and mRNAs using a SYBR Green PCR kit (TransGen, Beijing, China) according to the manufacturer's instructions. PCR amplification was performed using the following thermocycler program: denaturation at 95°C for 10 min; and 40 cycles of 95°C for 30 s, 58–60°C for 30 s, and 72°C for 30 s The PCR assays were performed using a CFX Connect Real-Time System (Bio-Rad, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference for mRNA expression, and U6 snRNA was used as an internal reference for lncRNA and miRNA expression. The primer sequences are listed in Table 1 . The relative quantitative expression levels of the genes were analyzed using the 2−ΔΔCt method. All experiments were performed in triplicate.
9
Quantifying H9N2 Viral RNA in Geese
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H9N2 viral RNA concentration was measured from fallopian tube and ovarian tissues in breeder goose infected with H9N2 AIV at 1, 3, 5, and 7 dpi using quantitative SYBR Green I real-time PCR (qRT-PCR) with the 7500 Fast Real-Time PCR System (Applied Bio systems, CA, USA). The primers were designed following conserved sequences of H9N2 strains in GenBank (Table 1 ). For confirming H9N2 AIV copy numbers in the above tissues, viral RNA concentration (log10) were normalized per 1 μg of total RNA. Furthermore, qRT-PCR was performed using the SYBR Green PCR kit (TransGen, Beijing, China) in a total volume of 25 μl following the product instructions. The amplification program was set as follows: 95°C for 5 min; 40 cycles of denaturation at 94°C for 15 s and 60°C for 34 s; and a final melting curve analysis step. Each sample was analyzed in triplicates.
10
Quantitative Gene Expression Analysis
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Total RNA was extracted from cell pellets using E.Z.N.A.® Total RNA Kit I (Omega Bio-Tek, Norcross, GA, USA) and reversely transcribed into cDNA using PrimeScript™ RT-PCR Kit (Takara Biotechnology, Dalian, China) according to the manufacturer's instructions. The target gene expressions were determined by qRT-PCR using specific primers of target genes (Table 2 ). GAPDH was served as an internal control for total cDNA content. For qRT-PCR analysis, aliquots of double-stranded cDNA were amplified using a SYBR Green PCR Kit (TransGen Biotech, Beijing, China). The cycling parameters were 45 cycles of 95°C for 15 seconds, 57°C for 15 seconds, and 72°C for 15 seconds. A melting curve analysis was then performed. The Ct was measured during the exponential amplification phase, and the amplification plots were analyzed using CFX Manager 2.1 software (Bio-Rad Laboratories, Hercules, CA, USA). The relative expression level (defined as fold change) of the target gene was determined by the following equation: 2−ΔΔCt (ΔCt = ΔCttarget – ΔCtGAPDH; ΔΔCt = ΔCtexpressing vector – ΔCtcontrol vector). The expression level was normalized to the fold change detected in the corresponding control cells, which was defined as 1.0.
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