ILC2Ps from WT, Il33−/−, and St2−/− mice were enriched for Lin-negative cells by magnetic separation and purified by FACS as described in Flow cytometry. Cells were sorted into cold PBS and centrifuged at 10,000 g for 5 min to remove the supernatant. mRNA was collected from the pelleted ILC2Ps using the RNeasy Micro kit (74004; QIAGEN) with on-column DNase treatment. cDNA was prepared using the SuperScript IV reverse transcription system (18091050; Invitrogen). Because of low cell input (fewer than 20,000 sorted ILC2Ps per sample), we performed a preamplification for our target genes as per manufacturer instructions (4384267; Applied Biosystems). A conventional quantitative PCR using the preamplification product and FAM-MGB TaqMan primers listed in Table S2 was performed on an Applied Biosystems QuantStudio12k Flex Real-Time PCR machine, and data were analyzed using Applied Biosystems QuantStudio 12k Flex Software v.1.2.2. Six candidate housekeeping genes were evaluated for stable expression in ILC2Ps as previously described (Vandesompele et al., 2002 (link)), and target genes were normalized to the geometric mean Ct values of a pool of three of these stably expressed reference genes: Rpl13a, Ywhaz, and Hprt. Differential expression was assessed by using the ΔΔCt protocol.
Quantstudio 12k flex software v 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio 12k Flex Software v.1.2.2 is a software suite designed to operate the QuantStudio 12k Flex Real-Time PCR System, a high-throughput genetic analysis platform. The software provides the necessary tools to control the instrument, collect and analyze data from real-time PCR experiments.
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Lab products found in correlation
Quantstudio12k flex real time pcr machine, by Thermo Fisher Scientific (3 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Hot firepol evagreen qpcr supermix, by Solis BioDyne (2 mentions)
Superscript 4 reverse transcription system, by Thermo Fisher Scientific (1 mentions)
Firepol evagreen qpcr supermix, by Solis BioDyne (1 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Dnase, by Thermo Fisher Scientific (1 mentions)
Superscript 4 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Quantstudio 12k flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Tempus spin rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Tempus blood rna tube, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Quantstudio 12k flex system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Molecular Research Center (1 mentions)
Recoverall total nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
Gel doc ez system, by Bio-Rad (1 mentions)
10 protocols using quantstudio 12k flex software v 1
1
Transcriptional Profiling of Murine ILC2Ps
2
Quantitative PCR for Mitochondrial DNA
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Every reaction was made in four parallel samples to minimize possible errors. All reactions were performed in a final volume of 10 μL. qPCR was carried out using primers obtained from TAG Copenhagen (Table S1 ) and HOT FIREPol EvaGreen qPCR Supermix (08-36-00001, Solis BioDyne, Tartu, Estonia). Expression level of HPRT was used as an internal reference. For mitochondrial:nuclear DNA ratio measurement, mitochondrial target was the gene for mt-CytB and nuclear target was a region of H4C. Real-time qPCR reactions were run on QuantStudio 12 K Flex Software v.1.2.2 Real-Time PCR System equipment (Applied Biosystems, Waltham, MA, USA) and quantified with the QuantStudio 12 K Flex Software v.1.2.2.
3
Quantitative Real-Time PCR Analysis
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Total RNAs were extracted from the OB and rostral FC using TRI Reagent®® (TR 118) (Molecular Research Center, Inc., Cincinnati, OH, USA), and cDNAs were synthesized with a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). Gene primers (Supplementary Table S1 ) designed in Primer3 with BLAST sequence verification (TAG Copenhagen AS, Frederiksberg, Denmark) were mixed with 5x HOT FIREPol®® EvaGreen®® qPCR Supermix (Solis BioDyne, Tartu, Estonia) and RT-qPCR was performed on equipment with QuantStudio 12KFlex Software v.1.2.2 (Applied Biosystems, Waltham, MA, USA) according to the respective manufacturer’s instructions. Quantification was performed by first normalizing Ct values of target genes to the reference gene (Gapdh), and mRNA levels were expressed as exponential fold-changes against the smallest ΔCt value (2−ΔΔCt).
4
Quantifying Il23p19 and Il33 Expression
5
Quantitative PCR Analysis of Il33 Isoforms
6
Quantitative Gene Expression Analysis Protocol
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For quantitative gene-expression analysis, total RNA of patient, her brother, parents, and ten healthy controls, was collected using Tempus Blood RNA tubes (Thermo Fisher Scientific), isolated using the Tempus Spin RNA Isolation kit (Thermo Fisher Scientific), and reverse-transcribed using the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific). RT-qPCR was performed using a QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific). The amounts of mRNA were calculated using the 2−ΔΔCt method, normalized against housekeeping genes GAPDH and TBP. RT-qPCR reactions were carried out using the TaqMan method (TaqMan ID# Hs00430773_m1 RAI1 NM_030665 ex 2-3, Hs01554690_m1 RAI1 NM_030665 ex 3-4, Hs99999905_m1 GAPDH; Hs00427620_m1 TBP), and SYBR Green methodology (Primers details are reported in Supplementary Table S2 ). Data were analyzed using the QuantStudio 12K Flex Software v1.2.3 (Thermo Fisher Scientific). We established the proper range of gene expression in ten healthy controls by calculating the mean value ±2 standard deviation.
7
Real-time PCR Analysis of Macrophage Polarization
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The primers (Table S4 ) were designed using Primer‐Blast tool19 and evaluated with Beacon Designer Free Edition (Premier Biosoft, Palo Alto, CA, USA). Real‐time PCR (rtPCR) was performed using the Solis Biodyne Supermix (Solis Biodyne, Tartu, Estonia) in accordance with manufacturer's recommendations. rtPCR was performed on a QuantStudio 12K Flex system (Thermo Fisher Scientific) (Data S1 ).
The 2−ΔΔCt analysis was performed using the QuantStudio 12k Flex Software v1.2.3 (Thermo Fisher Scientific) to obtain the cycle of threshold (Ct) for each sample investigated. Actin beta (ACTB) was used as reference gene. The mean fold change expression was calculated for M2a, M2a + IgG1, and M2a + IgG4.
The 2−ΔΔCt analysis was performed using the QuantStudio 12k Flex Software v1.2.3 (Thermo Fisher Scientific) to obtain the cycle of threshold (Ct) for each sample investigated. Actin beta (ACTB) was used as reference gene. The mean fold change expression was calculated for M2a, M2a + IgG1, and M2a + IgG4.
8
RT-qPCR Analysis of Gene Expression
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Total RNAs were extracted from the PFC tissue by TRIzol (Molecular Research Center, Cincinnati, OH, United States) and reversely transcribed with a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waitham, MA, United States). RT-QPCR was performed by using corresponding primers and 5 × HOT FIREPol® EvaGreen® qPCR Supermix (Solis BioDyne, Tartu, Estonia) on a PCR instrument equipped with QuantStudio 12KFlex Software v.1.2.2 (Thermo Fisher Scientific, Waitham, MA, United States) according to the instructions of the respective manufacturers. The primers (Ma et al., 2020 (link)) listed in Supplementary Table 1 were purchased from TAG Copenhagen A/S (Frederiksberg, Denmark). Quantification was performed by normalizing Ct values of target genes to that of the reference gene (β-actin) and expressed as exponential fold changes against the average ΔCt value of the control group (2–ΔΔCt).
9
PAX8/PPARG Fusion Detection in FFPE
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RNA was isolated from three 10-μm-thick FFPE sections using the Recover All Total Nucleic Acid Isolation kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Total RNA (500 ng) was reverse transcribed into cDNA, and quality was determined by amplification of the internal control gene (RPS8) as previously described (38 (link)). Screening for PAX8/PPARG rearrangement (fusion of exon 9 of PAX8 and exon 2 of PPARG) was performed by quantitative RT-PCR. Briefly, cDNA was synthesized as previously described (39 (link)) and subjected to PCR amplification using the TaqMan assay (Assay ID Hs04396714_ft, Thermo Fisher Scientific). The quantitative PCR reaction was performed in triplicate, and the threshold cycle was obtained using QuantStudio™ 12K Flex Software v1.2.2 (Thermo Fisher Scientific). PCR products were analyzed by electrophoresis on a 2% agarose gel and visualized on a Bio-Rad Gel Doc EZ system (Bio-Rad).
10
Quantitative Real-Time PCR Protocol
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Total RNA was isolated using the TRNzol reagent (TIANGEN, cat: DP424) and converted to cDNA using the Fast King RT Kit (TIANGEN, cat: KR116) according to the manufacturer’s instructions. Real-time quantitative PCR was carried out in a 10 μl reaction containing the SYBR Select Master Mix (Vazyme, cat: Q331) in technical quadruplicate using a Quantstudio 6K Flex system (Life Technologies). Results were analyzed using the Quantstudio 12 K Flex Software v1.2.2 (Thermo Fisher Scientific). Relative mRNA levels were calculated based on the 2−ΔΔCT method, using the 18S rRNA as references.
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