Total RNA was extracted using RNeasy Plus Mini kit (QIAGEN, 74136). cDNA libraries were generated using Trio RNA-Seq kit (Tecan, 0506). For RT-PCR, cDNA was generated using SuperScript IV First-Strand Synthesis System (Thermo Fisher, 18091050). RT-PCR was performed in technical triplicate using SYBR Green PCR Master mix (Applied Biosystems, 4309155) and 0.25 μM concentration of each primer on a QuantStudio 12K-flex machine (Life Technologies). Fold change with respect to GAPDH was calculated using the ddCt method. A linear range was confirmed for each primer pair by a five-fold serial dilution curve. Primer sequences are listed below. For RNA sequencing, 75 bp paired-end reads were generated by Illumina NextSeq 500.
Quantstudio 12k flex machine
Manufactured by Thermo Fisher Scientific
The QuantStudio 12K Flex is a high-throughput real-time PCR system capable of running 12 independent samples simultaneously. It is designed to perform a variety of real-time PCR applications, including gene expression analysis, genotyping, and high-throughput screening. The instrument features a flexible block design, allowing for the use of multiple sample formats from 96-well to 384-well plates. The QuantStudio 12K Flex is compatible with a wide range of real-time PCR chemistries and supports advanced data analysis tools.
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Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Superscript 4 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Trio rna seq kit, by Tecan (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Taqman openarray mouse inflammation panel, by Thermo Fisher Scientific (1 mentions)
Purelink rna purification kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Superscript 3 first strand synthesis supermix, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
7 protocols using quantstudio 12k flex machine
1
RNA Extraction, cDNA Synthesis, and qRT-PCR
2
Profiling Inflammation Genes in Keratinocytes
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Cultured keratinocytes in one well of a 6-well plate were lysed in 500 μl TRIzol (Invitrogen) and the RNA was purified using PureLink RNA purification kit (Ambion, Carlsbad, CA). cDNA was made with SuperScript VILO cDNA Synthesis kit (Invitrogen) and qPCR was performed using TaqMan Open Array mouse inflammation panel (Applied Biosystems, Carlsbad, CA) on a QuantStudio 12K Flex machine (Life Technologies) following the manufacturer protocols.
3
SEA-induced Immune Checkpoint Regulation
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To measure mRNA expression of various genes before and after SEA activation, human and cynomolgus monkey PBMCs were treated with 20 ng/mL SEA for 6 days. Naive control PBMCs (untreated with SEA) and SEA-treated PBMCs were harvested and RNA was extracted from these samples with RNeasy mini kit (Qiagen, 74106). Synthesis of cDNA was done with SuperScript VILO cDNA Synthesis Kit (Life Technologies, 11754–050). Taqman PCR with primers (Life Technologies, 4370048) of β-actin (ACTB; Mf04354341_g1), TIM-3 (Mf02850235_m1), LAG-3 (Mf02841705_g1), PD-L1 (Mf02865485_m1), TIGIT (Mf02887844_m1) was run on QuantStudio 12 K Flex machine (Life Technologies). Using β-actin as the reference gene, relative gene expression was calculated as the level of mRNA expression of individual genes relative to that of β-actin which was set to 1. Calculations were done as follows: ΔCt = CtAvg GOI - CtAvg ACTB, where ACTB is the reference gene, GOI is gene of interest (e.g., TIM-3, LAG-3, PD-L1, TIGIT), and relative gene expression = 2ΔCt.
4
Quantifying Gene Expression in Human Tissues
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qPCR experiments were performed as previously described. Briefly, human tissue samples were ground using mortar and pestle in liquid nitrogen, and RNA was isolated using RNeasy kit (Qiagen) and converted to cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher). We performed qPCR with a Taqman Gene Expression Assay using a QuantStudio 12K Flex machine (Applied Biosystems). Primers were obtained from ThermoFisher for Actin (Catalog #4331182) and G6PD (Catalog #4331182).
5
Quantitative RT-PCR analysis of gene expression
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Total RNA was extracted from 30 to 40 dissected livers for each condition using the RNeasy Micro Kit (Qiagen, Hilden, Germany); cDNA was synthesized from the RNA using the SuperScript® III First-Strand Synthesis SuperMix (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocols. qRT-PCR was performed as previously described14 (link) using the QuantStudio 12K Flex machine (Applied Biosystems, Waltham, MA) with the iTaqTM Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA). eef1a1l1 was used for normalization as previously described60 (link). At least three independent experiments were performed. For each replicate with larval livers, the control value was first set to 1, and then the values of the experimental groups were calculated relative to the control value. The primers used for qRT-PCR are listed in Table S3 ; the Ct values of qRT-PCR data are listed in Table S4 .
6
Quantifying Gene Expression in Human Tissues
7
Quantitative RT-PCR Gene Expression Analysis
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cDNA was synthesized from approximately 1 μg of total RNA using First strand synthesis Reverse Transcriptase kit with random oligos (Invitrogen). Quantitative RT-PCR was performed using SYBR-Select Master mix (Applied Biosystems) with primers listed in the S1 Table , according to the protocol recommended by the manufacturer on a Quant Studio 12 K Flex machine. The PCR conditions consisted of an initial denaturation step at 95°C for 10 min followed by 40 cycles at 95°C for 15 s (denaturation) and 60°C for 1 min (elongation). The fold-changes were calculated relative to internal control eef1a1 expression. A minimum of three biological and technical replicas were used to generate SEM.
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