Total RNA was extracted from cultured cells and tissues using TRIzol® RNA Isolation Reagents (Invitrogen, Carlsbad, CA, USA). The expression of miR-222-3p was quantified by qRT-PCR using TaqMan microRNA assays (Applied Biosystems, Carlsbad, CA, USA) and normalized to endogenous control U6B. All the reagents were purchased from Applied Biosystems (Foster City, CA, USA). Mature miRNA was reverse-transcribed from total RNA using specific miRNA RT-primers in TaqMan microRNA assays with reagents in the TaqMan MicroRNA Reverse Transcription kit. qRT-PCR was performed using TaqMan MicroRNA Assay primers with the TaqMan Universal PCR Master Mix and analyzed with an ABI Prism 7000 Sequence Detection System (Applied Biosystems).The cDNA was generated using Prime Script RT Reagent Kit (TaKaRa; Dalian, PRC). For quantification of ERα, pS2, PR, cyclin D1, qRT-PCR was carried out on ABI Prism 7000 Sequence Detection System (Applied Biosystems) with SYBR Premix Ex Taq (TaKaRa; Dalian, PRC). The primer sequences are listed in Table 2 . For all the qRT-PCR experiments, values on the y-axis equal to 2−ΔCt, where ΔCt is the difference between gene Ct and normalizer gene Ct. Ct represents the threshold cycle at which fluorescence rises statistically significantly above the baseline. Gene expression data were obtained in triplicate in three independent experiments.
Abi prism 7000 sequence detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, Germany, China, Switzerland, Canada, Singapore, France, United Kingdom
The ABI Prism 7000 Sequence Detection System is a real-time PCR instrument designed for gene expression analysis and quantification. The system utilizes fluorescence-based detection technology to monitor the amplification of DNA samples in real-time during the PCR process.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (118 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (78 mentions)
Rneasy mini kit, by Qiagen (68 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (50 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (48 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (45 mentions)
Trizol, by Thermo Fisher Scientific (44 mentions)
Rneasy plus mini kit, by Qiagen (17 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (17 mentions)
Taqman pcr master mix, by Thermo Fisher Scientific (16 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (15 mentions)
Isogen, by Nippon Gene (15 mentions)
Primescript rt reagent kit, by Takara Bio (15 mentions)
Rneasy kit, by Qiagen (14 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (13 mentions)
Quantitect reverse transcription kit, by Qiagen (13 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (13 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (13 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (12 mentions)
Iscript cdna synthesis kit, by Bio-Rad (12 mentions)
710 protocols using abi prism 7000 sequence detection system
1
Quantification of miRNA and mRNA Expression
2
Quantitative Real-Time PCR Analysis of TMPRSS2-ERG Fusion Gene
3
RNA Extraction and qRT-PCR for NDV Detection
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RNA was extracted from the urine, fecal and throat samples by adding 60 μl sample to 90 μl Magnapure 96 external lysis buffer (6374913001, Roche Diagnostics, The Netherlands) as described before (Richard et al., 2020 (link)). Subsequently, the lysed sample was added to 60 μl Agencourt AMPure XP magnetic beads (A63880, Beckman Coulter, The Netherlands) and incubated 15 min at room temperature. Magnetic beads were washed three times with 70 % ethanol using the DynaMag-96 magnet (12027, Invitrogen, The Netherlands) and subsequently air-dried. RNA was eluted by 6 min of incubation in bidest H2O. NDV-specific quantitative reverse transcription-PCR was performed using 5 μl RNA in an ABI PRISM 7000 Sequence Detection System using TaqMan Fast Virus 1-Step Master Mix (both from Thermo Fischer) in a total volume of 30 μl. The NDV-specific primers used were described by Wise et al. (2004) (link). The reverse transcriptase step was 5 min at 50 °C, followed by 95 °C for 20 s. Cycling consisted of 40 cycles of 3 s denaturation at 95 °C, 5 s annealing at 54 °C and 31 s extension at 60 °C.
4
RNA Extraction and qRT-PCR Analysis of Leishmania-Infected Mouse Spleens
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RNA from the spleens from L. donovani-infected mice was extracted using TRIzol (Thermo Fisher Scientific) and cDNA was synthesized by reverse transcription. The spleens were homogenized with 1 ml TRIzol and φ1.0 stainless steel beads in the 2 ml tube using Micro Smash MS100R (Tomy Seiko) at 4°C and RNA was purified according to the manufacture’s protocol. The concentration of total RNA was measured by DU 730 Life Science UV/vis spectrophotometer (Beckman Coulter). A mixture of 1.25 μM oligo (dT)16, 0.5mM dNTPs (Thermo Fisher Scientific) and 1 μg of total RNA in a tube was incubated for 5 min at 65°C. After adding 5× first strand buffer and 10 mM DTT (Thermo Fisher Scientific), 200 U M-MLV (Thermo Fisher Scientific) was added and the tube was incubated for 50 min at 37°C and 15 min at 70°C. Real-time PCR assay was carried out using 2 μl of cDNA as the template, 10 μl of SYBR Select Master Mix (Thermo Fisher Scientific) and primers listed in S1 Table [11 (link),37 (link)] on the ABI Prism 7000 Sequence Detection System (Thermo Fisher Scientific). Data was analyzed by 2−ΔΔCt methods and normalized by GAPDH. The thermal cycling conditions for the PCR were 94°C for 10 min, followed by 40 cycles of 94°C for 15 sec and 60°C for 1 min.
5
Quantifying Gene Expression from Brain Samples
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Total RNA was isolated from whole brain by using the RNeasy Lipid Tissue Mini Kit or RNeasy Plus Universal Mini Kit (Qiagen, Hilgen, Germany). Complementary DNA was synthesized by using the Omniscript RT Kit (Qiagen) or SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA). Real-time PCR to determine levels of gal expression was performed either on the ABI Prism 7000 Sequence Detection System using the Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) or on the LightCycler 480 System II using the LightCycler 480 SYBR Green I Master (Roche Diagnostics). For every reaction, melting curve analysis was conducted to ensure that a single amplicon was produced in each sample. Levels of β-actin (actb) expression in each sample were used for normalization. The primers used for real-time PCR are listed in Supplementary file 3 .
6
Daphnia RNA Extraction and qRT-PCR
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At the end of experiments, 3–4 daphnids per treatment were stored in 100 μL RNAlater (Invitrogen, Carlsbad, CA) in 1.7 mL tubes. Samples were stored at 4 °C for 24 hours then transferred to −80 °C until RNA extraction. Daphnids were homogenized using a Bullet Blender (Next Advance, Inc, Troy, NY), and RNA was isolated using the SV Total RNA Isolation System (Promega, Madison, WI). RNA concentration was determined by measuring absorbance at 260 nm using Nanodrop-1000 spectrophotometer (ThermoFisher). cDNA was prepared from extracted RNA using ImProm-II™ Reverse Transcription System (Promega) with oligo (dT) primers.
mRNA levels were measured by quantitative RT-PCR. Preparation of primers for D. magna actin, gapdh, Dhb1, Dhb2, and EcR-A (Fig.6c ) have been described previously32 (link),41 (link). Actin and gapdh were used to normalize Dhb1, Dhb2, and EcR-A mRNA levels. PCR was performed using the ABI PRISM ® 7000 Sequence Detection System with SYBR®Green PCR Mastermix (ThermoFisher) or iTaq Universal Sybr Green Supermix (BioRad, Hercules, CA) in 96-well plates (Olympus Plastics, Genesee Scientific, San Diego, CA) sealed with ThermalSeal (Excel Scientific, Inc., Victorville, CA). mRNA levels were calculated from raw data using Genex software from BioRad which utilizes algorithms to normalize mRNA levels to multiple housekeeping genes42 (link).
mRNA levels were measured by quantitative RT-PCR. Preparation of primers for D. magna actin, gapdh, Dhb1, Dhb2, and EcR-A (Fig.
7
Validated Gene Expression Assays for Notch Pathway
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Validated Gene Expression Assays for human Jag1 (Hs01070032_m1), Hes1 (Hs00172878_m1), Hes4 (Hs00368353_g1) and β-Actin (ID: 4333762T) were purchased from ThermoFisher Scientific. PCR reactions were performed with 100 ng of cDNA on the whole sample collection and cell lines, using an ABI Prism 7000 Sequence Detection System and TaqMan Universal PCR Master Mix (ThermoFisher Scientific). Cycling conditions were: 10 min of denaturation at 95 °C and 40 cycles at 95 °C for 15 s and at 60 °C for 1 min. Quantitative values were calculated by using the PE Biosystems Analysis software and expressed as N target (NT). NT=2−ΔCt, wherein ΔCt value of each sample was calculated by subtracting the average Ct value of the target gene from the average Ct value of the β-Actin gene.
8
RNA Quantification via RT-qPCR
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RNA primers (primers used are exhibited in Table 2 ) were designed and colligated by Sangon Biotech (Shanghai, China). The total RNA of cells was extracted using Trizol. Next, the concentration and purity of the total RNA were measured in the same way. Total RNA were reverse‐transcribed into cRNA using a First Strand Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer guidelines. First strand cDNA was stored at −20°C before real‐time PCR, which subjected cDNA to the ABI PRISM 7000 Sequence Detection System using Maxima SYBR Green qPCR Master Mix (2×) (Thermo Fisher Scientific). The samples were amplified using β‐actin as internal control and reaction conditions were as follows: original denaturation at 95°C for 5 minutes with another extension at 72°C for 5 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds. After all of this was completed, results were analyzed using Step One Software v2.1 in 2−ΔΔCt, which analyzed the gene relative expression level.
9
Quantification of miR-141 Expression
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Total RNA was isolated from clinical samples using a TRIzol kit (Thermo Fisher Scientific, Inc.) and purified using a QiaQuick PCR Purification kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturers' protocols. The quantity of extracted RNA was examined by a NanoDrop-3000 spectrophotometer (Thermo Fisher Scientific, Inc.). A total of 5 µg RNA from each sample was reverse synthesized to complementary DNA using a TaqMan MicroRNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Gene expression of human miR-141 (hsa-miR-141) was quantified through RT-qPCR using a TaqMan MicroRNA assay kit (Thermo Fisher Scientific, Inc.) on an ABI Prism 7000 Sequence Detection system (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The thermocycling conditions used were as follows: 30 sec at 95°C; 30 sec at 60°C; and 30 sec at 72°C for 35 cycles. The following primers were used: hsa-miR-141 forward, 5′-CGCTAACACTGTCTGGTAAAG-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; U6 snRNA forward, 5′-ATTGGAACGATACAGAGAAGATT-3′ and reverse, 5′-GGAACGCTTCACGAATTTG-3′. The relative expression of hsa-miR-141 was measured as a fold-change and was normalized to U6 small nuclear RNA expression in control samples using the 2−ΔΔCq method (22 (link)).
10
Analyzing Inflammatory Cytokines in Mouse Footpads
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Mice footpads were homogenized with 1 ml TRIzol (Thermo Fisher Scientific) and φ1.0 stainless steel beads in the 2 ml tube using Micro Smash MS100R (TOMY, Tokyo, Japan) at 4°C using tissue homogenizer (Tomy, Micro Smash MS-100). RNA was isolated using TRIzol Reagent according to manufacturer instructions. RNA yield was measured by a spectrophotometer (Beckman Coulter DU-730). We found 30–53 μg RNA from each sample, and 2 μg of total RNA was used as the template for the synthesis of 20 μl cDNA. Synthesized cDNA was analyzed for mouse TNF-α, IL-1β, IL-6, IL-12 p40, inducible nitric oxide synthase (iNOS), and arginase 1 by reverse transcriptase real-time PCR. PCR method and primer sequences were described before [13 (link)]. Briefly real-time polymerase chain reaction (PCR) assay was carried out using 1 μl of cDNA as the template, 10 μl of SYBR Select Master Mix (Thermo Fisher Scientific) and primers on the ABI Prism 7000 Sequence Detection System (Thermo Fisher Scientific). Data were analyzed by 2−ΔΔ Ct methods and normalized by GAPDH. The thermal cycling conditions for the PCR were 94°C for 10 min, followed by 40 cycles of 94°C for 15 s and 60°C for 1 min.
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