Total RNA was extracted using a Trizol reagent (Invitrogen). RNA was treated with RNase-free DNase and purified using RNase mini kit (Qiagen). Reverse transcription reaction was performed with 500ng of total RNA using RT2-first strand kit (SABioscience). Rat mitochondrial energy metabolism PCR array was performed to measure gene expression of the electron transport chain and oxidative phosphorylation complexes (Rat mitochondrial energy metabolism, PARN-008ZD. SABioscience) by using Bio-Rad CFX 96 touch real-time PCR detection system.
Cfx 96 touch real
Manufactured by Bio-Rad
Sourced in United States
The CFX 96 Touch Real-Time PCR Detection System is a compact, high-performance instrument designed for real-time PCR applications. It features a 96-well format and supports multiple fluorescence detection channels for a wide range of experimental needs.
Automatically generated - may contain errors
Lab products found in correlation
Rnase mini kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nanodrop one spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Genelute mammalian total rna miniprep kit, by Merck Group (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Kapa sybr fast qpcr kit, by Roche (1 mentions)
Ampure xp, by Beckman Coulter (1 mentions)
4 protocols using cfx 96 touch real
1
Mitochondrial Energy Metabolism Profiling
2
Quantifying Gene Expression with RT-qPCR
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RNA was extracted and isolated using the GenElute Mammalian Total RNA Miniprep Kit and associated DNase digestion kit (RTN 350; MilliporeSigma). A NanoDrop One spectrophotometer (Thermo Fisher Scientific) was used to validate RNA quality and quantity. Total RNA was reverse transcribed using the iScript cDNA synthesis kit (1708891; Bio-Rad Laboratories). RT-qPCR was performed using iQSYBR Green Supermix (1725121; Bio-Rad Laboratories) in a Bio-Rad CFX96 Touch real-time PCR machine according to the manufacturer's instructions. Gene expression was calculated using the comparative cycle threshold method and normalized to human β-actin. Primers used were as follows: actin (5′-ACTGGGACGACATGGAGAAAA-3′; 5′-GCCACACGCAGCTC-3′), caspase-1 (5′-CAACTACAGAAGAGTTGAGG-3′; 5′-AACATTATCTGGTGTGGAAG-3′), IL-1β (5′-CTAAACAGATGAAGTGCT-3′; 5′-GGTCATTCTCCTGGAAG-3′), and NLRP3 (5′-AGGTGTTGGAATTAGACAAC-3′; 5′-AATACATTTCAGACAACCCC-3′). All sample processing was performed in duplicate, and each experiment was performed with at least three biological replicates. Heatmaps based on RNA-seq log2 reads per kilobase million values were generated using previously published datasets available in the Sequence Read Archive (SRP067137, SRP109039).
3
Quantitative Gene Expression Analysis
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Total RNA was extracted using Trizol. Two micrograms of total RNA was transcribed into cDNA using the SuperScript™ III First-Strand Synthesis System (Invitrogen). qPCR was performed on a CFX96 Touch™ real-time PCR detection system (Bio-Rad Laboratories) using the SYBR green method based on the manufacturer’s manual. Gene-specific primers are listed in Table S1 in Additional file 1 . Gene expression was normalized to GAPDH and the relative gene expression was calculated using the 2-ΔΔCt method [28 (link)].
4
Quantitative 16S rRNA Gene Analysis
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For the measurement of the bacterial abundance, quantitative PCR (qPCR) was performed using the extracted DNA, diluted 50 times with nuclease-free water. The 515F/806R primer pairs described above were used to amplify the V4 region of the 16S rRNA. For the standard curve, the PCR products from the DNA extracted from the control pots were used, which were purified with AMPure XP (Beckman Coulter) and further diluted to 5 stages of different concentrations. Samples were prepared with 10.4 μL of KAPA SYBR Fast qPCR kit (Kapa Biosystem, USA), 0.08 μL of the forward primer, 0.08 μL of the reverse primer, and 2 μL of diluted DNA extract. Moreover, nuclease-free water was added to achieve the final volume of 20 μL. CFX96 Touch™ Real (Bio-Rad Laboratories, Inc., USA) was used and the cycling condition was set to 95°C for 30 s, 35 cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min, followed by 95°C for 1 min and subsequently 55°C to 95°C by 1°C increment for 10 s. The Ct values (Threshed cycle)
were calculated after the quantification of the amplification results using version 1.4.1 of the qR R package.
were calculated after the quantification of the amplification results using version 1.4.1 of the qR R package.
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