RNF5 expression was assessed using RT-qPCR. Total RNA was extracted from U251 cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse-transcribed into cDNA using the Quant One-Step RT-PCR kit (Tiangen Biotech Co., Ltd.). qPCR was performed using FastStart Universal SYBR Green Mix (Roche Diagnostics) and an ABI 7300 real-time PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers for RNF5 and β-actin were designed as follows: RNF5, forward 5′-GTACCCATACGATGTTCCAGATTACGC-3′, reverse 5′-CTGAGCAGCCAGAAAAAGAAAAAGATG-3′; and β-actin forward, 5′-CATGTACGTTGCTATCCAGGC-3′, and reverse, 5′-CGCTCGGTGAGGATCTTCATG-3′. Thermocycling conditions included pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec and extension at 72°C for 1 min for 35 cycles. Expression level of RFN5 was calculated using the 2−ΔΔCq method (17 (link)).
Fast start universal sybr green mix
Manufactured by Roche
Sourced in United States, Japan
Fast Start Universal SYBR Green Mix is a ready-to-use solution for real-time PCR applications. It contains all the necessary components, including a DNA polymerase, SYBR Green I dye, and buffers, for efficient amplification and detection of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (6 mentions)
Abi 7300 real time pcr instrument, by Thermo Fisher Scientific (3 mentions)
Dnase, by Thermo Fisher Scientific (3 mentions)
Cfx96 384 pcr detection system, by Bio-Rad (3 mentions)
Quant one step rt pcr kit, by Tiangen Biotech (2 mentions)
Verso cdna kit, by Thermo Fisher Scientific (2 mentions)
Stratagene mx3000p real time pcr system, by Agilent Technologies (2 mentions)
Tri reagent, by Molecular Research Center (2 mentions)
Rnaiso plus, by Takara Bio (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Quantstudio 3 instrument, by Thermo Fisher Scientific (1 mentions)
Cfx96 384, by Bio-Rad (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Abi 7500 real time pcr detection system, by Roche (1 mentions)
15 protocols using fast start universal sybr green mix
1
Quantitative Analysis of RNF5 Expression
2
Quantitative miRNA Expression Analysis
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MiRNA expression was assessed by qRT-PCR. Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse-transcribed using the Quant One-Step RT-PCR Kit (Tiangen, Beijing, China). PCR was performed using an ABI 7300 real-time PCR instrument (Applied Biosystems, Carlsbad, CA, USA) and FastStart Universal SYBR Green Mix (Roche, Basel, Switzerland). Expression levels were calculated using the 2−ΔΔCt method.
3
Sesamol Modulates Oxidant Enzymes
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DLD-1/COX-2-B2-βGal-BSD cells were seeded at a density of 2.0 × 105 cells in 24-well plates, and incubated with 50 and 100 µM sesamol for 48 h. Total RNA was isolated using TRIzol Reagent (Invitrogen, Grand Island, NY), treated with DNase (Invitrogen) and 1 µg aliquots in a final volume of 20 µl were used for synthesis of cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Carlsbad, CA). Real-time polymerase chain reaction (PCR) was carried out using Fast Start Universal SYBR Green Mix (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Primers for human NOX1 (5’-CCA CTG TAG GCG CCC TAA GTT and 5’-ATG ACC GGT GCA AGG ATC C), NOX2 (5’-GCC CAA AGG TGT CCA AGC T and 5’-TCC CCA ACG ATG CGG ATA T), p22phox (5’-ACC GCC GTG GTG AAG CT and 5’-ACC GAG AGC AGG AGA TGC A), GAPDH (5'-CCA CCC ATG GCA AAT TCC and 5'-TGG GAT TTC CAT TGA TGA CAA) were employed. Primers for mice NOX1 (5'-TCC CTT TGC TTC CTT CTT GA and 5'-CCA GCC AGT GAG GAA GAG TC), NOX2 (5'-GGG GTG TTG AAG GTC TCA AA and 5'-TGT TAC CAA CTG GGA CGA CA), p22phox (5'-CGT GGC TAC TGC TGG ACG TT and 5'-TGG ACC CCT TTT TCC TCT TT), GAPDH (5'-TTG TCT CCT GCG ACT TCA and 5'-CAC CAC CCT GTT GCT GTA) were employed. To assess the specificity of each primer set, amplicons generated from the PCR reaction were analyzed for melting curves.
4
Quantifying ac-AKH Expression in Aplysia
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To examine the pattern of ac-AKH expression, tissues were collected from control animals previously injected with 500 μL ASW (from Experiment 1). The central nervous system (CNS), heart, kidney, gill, esophagus, crop, and intestine were harvested, snap-frozen on dry ice, and stored at −70°C until RNA isolation. Total RNA was isolated using TRIzol reagent (Ambion, Austin, TX) according to the manufacturer's instructions. cDNA synthesis was performed from 1 μg total RNA using the QuantiTect Reverse Transcription Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions.
For quantitative reverse-transcription PCR (qPCR), A. californica actin (ac-actin) was used as a reference gene to normalize the target gene expression. qPCR primers for ac-akh were designed based on the previously published sequence (11 (link)). qPCR of ac-akh was carried out using FastStart Universal SYBR Green mix (Roche, Indianapolis, IN) and calculated using the 2−ΔΔCT method (26 (link)). Each reaction contained 9 μL SYBR Green mix, 9 μL ultrapure water, 0.5 μM gene-specific primers, and 2 μL (for ac-actin and ac-akh) of cDNA. Forward and reverse primers were 5′-CACACTGTCCCCATCTACGA and 5′-CCAGCGAGATCCAATCTCAT for ac-actin, and 5′-TTAATACAGCGAACCGCAAA, and 5′-TCACAGTTCTGGGCAGGTATT for ac-akh.
For quantitative reverse-transcription PCR (qPCR), A. californica actin (ac-actin) was used as a reference gene to normalize the target gene expression. qPCR primers for ac-akh were designed based on the previously published sequence (11 (link)). qPCR of ac-akh was carried out using FastStart Universal SYBR Green mix (Roche, Indianapolis, IN) and calculated using the 2−ΔΔCT method (26 (link)). Each reaction contained 9 μL SYBR Green mix, 9 μL ultrapure water, 0.5 μM gene-specific primers, and 2 μL (for ac-actin and ac-akh) of cDNA. Forward and reverse primers were 5′-CACACTGTCCCCATCTACGA and 5′-CCAGCGAGATCCAATCTCAT for ac-actin, and 5′-TTAATACAGCGAACCGCAAA, and 5′-TCACAGTTCTGGGCAGGTATT for ac-akh.
5
Quantitative Analysis of RNA Expression
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Total RNA from RCC tissues and cells was isolated using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). For mRNA, total RNAs were reversed with Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Basel, Switzerland). For miRNA, cDNA was synthesized using TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific). qPCR was conducted on an ABI 7300 real-time PCR instrument (Applied Biosystems; Thermo Fisher Scientific) with FastStart Universal SYBR® Green Mix (Roche Diagnostics).
The specific primers used in this study were listed as follows: E2F2 F: 5′-AGCTGGATCTGGAGGGGATT-3′, R: 5′-AGGACCCCATCCTCTGACTC-3′; SPTLC1 F: 5′-GCCAGGGATACTGCTTTTCA-3′, R: 5′-TTTGTCCGCACTTTTCCTTC-3′; miR-16–5p F: 5′-TCCACTCTAGCAGCACGTAAAT-3′, R: 5′-TCACACTAAAGCAGCACAGTAAT-3′; U6 F: 5′-CTCGCTTCGGCAGCACA-3′, R: 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH F: 5′-CTGGGCTACACTGAGCACC-3′, R: 5′-AAGTGGTCGTTGAGGGCAATG-3′. GAPDH or U6 was utilized as internal controls. Expression levels were quantified using 2−∆∆Ct method.
The specific primers used in this study were listed as follows: E2F2 F: 5′-AGCTGGATCTGGAGGGGATT-3′, R: 5′-AGGACCCCATCCTCTGACTC-3′; SPTLC1 F: 5′-GCCAGGGATACTGCTTTTCA-3′, R: 5′-TTTGTCCGCACTTTTCCTTC-3′; miR-16–5p F: 5′-TCCACTCTAGCAGCACGTAAAT-3′, R: 5′-TCACACTAAAGCAGCACAGTAAT-3′; U6 F: 5′-CTCGCTTCGGCAGCACA-3′, R: 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH F: 5′-CTGGGCTACACTGAGCACC-3′, R: 5′-AAGTGGTCGTTGAGGGCAATG-3′. GAPDH or U6 was utilized as internal controls. Expression levels were quantified using 2−∆∆Ct method.
6
Quantitative Real-Time RT-PCR Analysis
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Total RNA was extracted by Trizol reagent (Thermo Fisher, Waltham, MA) following the manufacturer’s instructions. Total RNA (2 µg) was used to perform reverse transcription with Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher). Quantitative real-time RT-PCR analysis was performed with a Quantstudio3 instrument (Thermo Fisher) using Fast Start Universal SYBR Green Mix (Roche, Mannheim, Germany). The primers, synthesized by Sangon Biotech., Shanghai, China., and used for real-time RT-PCR were displayed in Table S2 . The method of 2−∆∆Ct was used to calculate the relative mRNA level. Data were normalized to GAPDH expression.
7
Intestinal Polyp RNA Extraction and qPCR
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Total RNA was isolated from intestinal polyps and non-polyp-containing intestinal mucosa using TRIzol Reagent (Invitrogen, Grand Island, NY, USA), treated with DNase (Invitrogen) and 1 μg in a final volume of 20 μL was used for cDNA synthesis using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, USA). Real-time PCR was conducted using a CFX96/384 (BIO RAD, Tokyo, Japan) and Fast Start Universal SYBR Green Mix (Roche Diagnostics, Mannheim, Germany) according to the manufacturers’ instructions. Primer sequences were as follows: IKBKB (5′-ATC AGG CGA CAG GTG AAC AG and 3′-GGC CAC AGC AGT TCT CGA A), IL-1β (5′-GAA ATG CCA CCT TTT GAC AGT G and 3′-TGG ATG CTC TCA TCA GGA CAG), IL-6 (5′-TGT TCT CTG GGA AAT CGT GGA and 3′-AAG TGC ATC ATC GTT GTT CAT ACA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5′-TTG TCT CCT GCG ACT TCA and 3′-CAC CAC CCT GTT GCT GTA). To assess the specificity of each primer set, amplicons generated from the PCR reactions were analyzed for melting curves.
8
Quantitative Real-Time PCR Analysis
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Total RNA from the cells was isolated using Tri Reagent (Molecular Research Center Inc., Cincinnati, OH, USA). The cDNAs were synthesized using the Verso cDNA kit (Thermo Scientific, San Jose, CA, USA). The quantitative real-time PCR was performed with Fast Start Universal SYBR Green mix (Roche Applied Science, Indianapolis, IN, USA) using the Stratagene Mx3000P real-time PCR system (Agilent Technologies, Santa Clara, CA, USA). The primer sequences were the same as used in (13 (link)). Relative mRNA expression levels were compared by using the 2−ΔΔC(T) method, with Ribosomal protein, Large, P0 (RPLP0) as reference gene.
9
Real-Time PCR Analysis of Gene Expression
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Total RNA was isolated using TRIzol (Invitrogen, Grand Island, NY, USA), and 1 µg aliquots in a final volume of 20 µL were used for cDNA synthesis using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR was carried out using the CFX96/384 PCR Detection System (Bio-Rad) and Fast Start Universal SYBR Green Mix (Roche Diagnostics, Mannheim, Germany) according to the manufacturers’ instructions. The primer sequences were as follows: 5′-GCCCGCGCCCAGACAGGATA, 3′-GCGGCGGCGGAGAGGA (c-Myc), 5′-CTGGTGTTTGAGCATGTAGACC, 3′-GATCCTTGATCGTTTCGGCTG (Cdk4), 5′-TTGTCTCCTGCGACTTCA, 3′-CACCACCCTGTTGCTGTA (GAPDH). To assess the specificity of each primer set, the melting curves of the amplicons generated by the PCR reactions were analyzed.
10
Gene Expression Analysis of Intestinal Samples
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Tissue samples from the proximal segment of small intestinal mucosa and polyps of mice were rapidly deep-frozen in liquid nitrogen and stored at −80 °C.
Total RNA was isolated from tissue samples by using RNAiso Plus (TaKaRa, Shiga, Japan); 100 ng aliquots in a final volume of 20 µL were used for synthesis of cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and oligo (dT) primers. Real-time PCR was carried out using the CFX96/384 PCR Detection System (BIO RAD, Tokyo, Japan) and Fast Start Universal SYBR Green Mix (Roche Diagnostics, Mannheim, Germany), according to the manufacturers’ instructions. The primer sequences were as follows: c-Myc (5′-GCTCGCCCAAATCCTGTACCT and 3′-TCTCCACAGACACCACATCAATTTC), cyclin D1 (5′-TGACTGCCGAGAAGTTGTGC and 3′-CTCATCCGCCTCTGGCATT) and GAPDH (5′-TTGTCTCCTGCGACTTCA and 3′-CACCACCCTGTTGCTGTA). To assess the specificity of each primer set, the melting curves of the amplicons generated by the PCR reactions were analyzed.
Total RNA was isolated from tissue samples by using RNAiso Plus (TaKaRa, Shiga, Japan); 100 ng aliquots in a final volume of 20 µL were used for synthesis of cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and oligo (dT) primers. Real-time PCR was carried out using the CFX96/384 PCR Detection System (BIO RAD, Tokyo, Japan) and Fast Start Universal SYBR Green Mix (Roche Diagnostics, Mannheim, Germany), according to the manufacturers’ instructions. The primer sequences were as follows: c-Myc (5′-GCTCGCCCAAATCCTGTACCT and 3′-TCTCCACAGACACCACATCAATTTC), cyclin D1 (5′-TGACTGCCGAGAAGTTGTGC and 3′-CTCATCCGCCTCTGGCATT) and GAPDH (5′-TTGTCTCCTGCGACTTCA and 3′-CACCACCCTGTTGCTGTA). To assess the specificity of each primer set, the melting curves of the amplicons generated by the PCR reactions were analyzed.
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