Total RNA was extracted from bursal tissue collected from the experimental birds of different groups at weekly intervals (six chicks from each group) by using RNeasy Mini Kit (Invitrogen, USA) according to the manufacturer’s protocol. The purity and concentration of the extracted RNA was checked on 260/280 nm by ScanDrop2 (Analytik Jena). Subsequently, 500 ng purified RNA was reverse transcribed to cDNA using Revertaid™ First Strand cDNA Synthesis Kit (Thermo Scientific, USA) as per the manufacturer’s protocol.
Scandrop2
Manufactured by Analytik Jena
Sourced in Germany
The ScanDrop2 is a compact and versatile spectrophotometer designed for a wide range of laboratory applications. It features a user-friendly touchscreen interface and a robust, durable build. The instrument is capable of performing accurate absorbance measurements across a wide wavelength range.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Thermo Fisher Scientific (1 mentions)
Milk bacterial dna isolation kit, by Norgen Biotek (1 mentions)
Nucelospin rna kit, by Macherey-Nagel (1 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Quantstudio 6, by Thermo Fisher Scientific (1 mentions)
Hiscript 2 q rt kit, by Vazyme (1 mentions)
Qtower3g real time pcr system, by Analytik Jena (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (1 mentions)
Easyspin plus polysaccharide polyphenol complex plant rna rapid extraction kit rn53, by Aidlab (1 mentions)
Evo m mlv rt kit, by Accurate Biology (1 mentions)
5 protocols using scandrop2
1
RNA Extraction and cDNA Synthesis
2
Optimized Milk Bacterial DNA Extraction
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The bacterial genomic DNA (gDNA) was extracted from 100 μL of milk tested to be sterile by culturing on LB broth. It was spiked with 108 CFU/mL of ATCC strains of the six bacteria for optimization of the developed assay. Milk Bacterial DNA Isolation Kit (Norgen Biotek Corp., ON, Canada) was used according to the protocol for unknown strain of bacteria. The dsDNA concentration and purity of all six bacterial DNA were measured using the Scandrop2 (Analytik Jena, Jena · Germany). Each bacterial species was confirmed by amplification using the primers listed in Table 1
3
Hyper-IL-6 Signaling and SOCS3 Expression
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Ba/F3-gp130 cells were washed 3 times with PBS and serum starved for 2.5 h. 5 nM Hyper-IL-6 or 5nM sIL-6R/sIL-11R plus 5 nM IL-6/IL-11 were incubated with 1 μg/mL sgp130Fc variant for 20 min. Then, the cells were stimulated with the mixture for 1 h. Cells were harvested and the RNA was isolated using the NuceloSpin RNA Kit (Macherey-Nagel, Düren, Germany) according to manufacturers' instructions. The RNA concentration was measured on a ScanDrop2 (Analytik Jena, Jena, Germany) and 2 μg RNA were reversely transcribed into cDNA to a final volume of 20 μL using 5 μM Oligo-dT primer and the RevertAid Reverse Transcriptase (Thermo Fisher Scientific, Waltham, USA) according to manufacturers' instructions. For quantitative PCR, cDNA transcribed from 50ng RNA was analyzed using the SYBR Green Mastermix (Thermo Fisher Scientific, Waltham, USA) in a total volume of 10μL according to manufacturers' instructions. The PCR was performed on a QuantStudio 6 (Thermo Fisher Scientific, Waltham, USA). The samples were initially incubated at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. The measurements were performed in triplicates. SOCS3 expression was normalized to GAPDH and the relative gene expression levels were calculated using the 2−ΔΔCt method.
4
Quantitative Real-Time PCR Gene Expression
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Total RNA was extracted from cell by using Trizol reagent (Sigma; T9424). The quantity and quality of RNA were determined using a ScanDrop2 nano-volume spectrophotometer (Analytik Jena), and reversely transcribed based on the HiScript II Q RT kit (Vazyme; R223) according to the manufacturer’s instructions. Amplification and real-time detection were performed on a qTOWER3 G real-time PCR system (Analytik Jena) by using ChamQ Universal SYBR qPCR Master Mix (Vazyme; Q711) in 20 μL reaction. The relative expression levels of each targeted gene were normalized by subtracting the corresponding mouse β-actin threshold cycle (CT) values by using the ΔΔCT comparative method. Three biological replicates per group were used for qPCR. Primers were synthesized by Sangon Biotech (Shanghai, China). Sequences of all primers used are provided in Additional file 1 : Table S3.
5
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted using the EASYspin Plus polysaccharide polyphenol complex plant RNA rapid extraction kit (RN53) (Aidlab, Beijing, China) according to the manufacturer's instructions. RNA integrity and concentration were checked by 1.0% agarose gel and a ScanDrop 2 nucleic acid detector (Analytik Jena, Jena, Germany), respectively. Qualified RNA was reverse-transcribed to cDNA using the Evo M-MLV RT Kit (AG11705) (Accurate Biology, Changsha, China).
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