Cell lysis, reverse transcription, and qPCR were performed with a SuperPrep Cell Lysis RT Kit for qPCR (TOYOBO) and Power SYBR Green PCR Master Mix (Life Technologies), according to the manufacturers’ instructions. Quantitative real-time PCR was performed with a Step-One-Plus PCR system (Applied Biosystems) by the ΔΔCT method. For analyses with mouse-derived immune cells, total mRNA was extracted with an RNeasy Mini Kit (Qiagen), and then transcribed to cDNA with ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO), according to the manufacturers’ instructions. Quantitative real-time PCR was performed with Power SYBR Green PCR Master Mix (Life Technologies) and a Step-One-Plus PCR system by the ΔΔCT method. Moreover, for the analysis with psoriasis-model mice, total mRNA was extracted by the same method used for the mouse immune cells. Quantitative real-time PCR was performed with TaqMan Gene Expression Master Mix (Applied Biosystems) and a Step-One-Plus PCR system, using the following TaqMan Gene Expression Assays. Primers used for this study are listed in Supplementary Table 2 .
Steponeplus pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, France, Japan, Germany, Canada
The StepOnePlus PCR System is a real-time PCR instrument designed for a variety of applications, including gene expression analysis, genotyping, and pathogen detection. It provides accurate and reliable results through its advanced optical system and thermal control technology.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (53 mentions)
Trizol, by Thermo Fisher Scientific (32 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (28 mentions)
Rneasy mini kit, by Qiagen (26 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (15 mentions)
Primescript rt reagent kit, by Takara Bio (14 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (11 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (9 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (9 mentions)
Miscript 2 rt kit, by Qiagen (8 mentions)
Sybr green master mix, by Thermo Fisher Scientific (8 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (8 mentions)
Nanodrop, by Thermo Fisher Scientific (8 mentions)
Sybr premix ex taq, by Takara Bio (8 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (8 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (7 mentions)
Sybr green pcr master mix, by Takara Bio (6 mentions)
Stepone software v2, by Thermo Fisher Scientific (6 mentions)
High pure rna isolation kit, by Roche (6 mentions)
Maxima h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (6 mentions)
278 protocols using steponeplus pcr system
1
Single-Cell Gene Expression Analysis
2
RT-qPCR Validation of miRNA and mRNA
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Reverse transcription-quantitative PCR (RT-qPCR) of miRs in 70 samples was carried out by a TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific, USA) and TaqMan Advanced miRNA Assays (Thermo Fisher Scientific, USA), in 96-well plates on the StepOnePlus PCR System (Applied Biosystems, USA), according to the manufacturer protocols. All samples were normalized to hsa-miR-191 levels and spike-in control cel-miR-39-3p. RT-qPCR assays were performed to validate the DESeq2 differential expression results for miRNAs with mean of normalized counts > 100.
A two-step qRT-PCR of mRNA in 70 samples was carried out using a High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, USA) and Custom TaqMan Array 48 Plus plates (Thermo Fisher Scientific, USA), in 96-well plates on the StepOnePlus PCR System (Applied Biosystems, USA), according to the manufacturer protocol. All samples were normalized to GUSB and levels of GAPDH, HPRT served as secondary internal controls.
Data analysis was performed by SDS software (version 2.3. Applied Biosystems). A P value < 0.05 was considered statistically significant. In order to account for multiple comparisons, a correction for the false discovery rate (Q values <0.10) was calculated using the Benjamini-Hochberg adjustment.
A two-step qRT-PCR of mRNA in 70 samples was carried out using a High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, USA) and Custom TaqMan Array 48 Plus plates (Thermo Fisher Scientific, USA), in 96-well plates on the StepOnePlus PCR System (Applied Biosystems, USA), according to the manufacturer protocol. All samples were normalized to GUSB and levels of GAPDH, HPRT served as secondary internal controls.
Data analysis was performed by SDS software (version 2.3. Applied Biosystems). A P value < 0.05 was considered statistically significant. In order to account for multiple comparisons, a correction for the false discovery rate (Q values <0.10) was calculated using the Benjamini-Hochberg adjustment.
3
Quantitative Expression Analysis of Lung Genes
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Total RNA was extracted from lung tissues using the RNA extraction kit (Qiagen, Hilden, Germany). qRT-PCR was performed utilizing the qRT-PCR kit (Thermo Fisher, United States) in the ABI StepOnePlus PCR system according to the manufacturer’s protocol. The ACTB mRNA expression level was employed as an internal control. The primers were designed as follows: SMAD4, forward, 5′-GTCATCCTGCTCACCAGATGTC-3′ and reverse, 5′-TGCTCAGACAGGCATCGTTAC-3′; HIF-1, forward, 5′-AGCAAGATCTCGGCGAAGC-3′ and reverse, 5′-ACCACCGGCATCCAGAAGT-3′; MAPK, forward, 5′-ACAGGCAGCGGAGACACCTA-3′ and reverse, 5′-GGGGAGGATGATCGAGACAC-3′; HRAS, forward, 5′-ATCCAGCTGATCCAGAACCAC-3′ and reverse, 5′-TCCCGCATGGCACTATACTC-3′; SOD1, forward, 5′-CAGAAGGCAAGCGGTGAAC-3′ and reverse, 5′-GAGGTCCTGCACTGGTACAGC-3′; AKT2, forward, 5′-TGCTGCCGCCAGTTCATA-3′ and reverse, 5′-GCAGGAGGCTCCTCGGATAC-3′; RAC1, forward, 5′-CAGATGCAGGCCATCAAGTG-3′ and reverse, 5′-GTCAAAGACGGTGGGGATGT-3′; P53, forward, 5′-CTCCCTCTGAGCCAGGAGAC-3′ and reverse, 5′-GACACTCGGAGGGCTTCACT-3′; ACTB, forward, 5′-TTCATGGATGCCACAGGATT-3′ and reverse, 5′-TGACGGCCAGGTCATCACTA-3′. The qRT-PCR results were analyzed and expressed as the relative mRNA expression of the CT (threshold cycle) value, which was then converted to fold changes.
4
Quantitative RT-PCR Protocol for Gene Expression Analysis
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Total RNA was extracted using TRIzol (Invitrogen), and RNA was then reverse‐transcribed using SuperScript First‐Strand cDNA System (Takara) according to the manufacturer's instructions. Quantitative RT‐PCR (qRT‐PCR) was performed using the SYBR Green PCR master mix (Takara) and the StepOnePlus PCR system (Thermo Fisher Scientific) according to the manufacturer's instructions. The housekeeping gene GAPDH was used as an endogenous control. The primer sequences are shown in Table S1 .
5
RNA Extraction and qPCR Analysis
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Total RNA was extracted by using RNA Extraction Kit (TIANGEN BIOTECH, DP419), and reverse transcribed to cDNA by using Prime Script RT Kit (Takara, RR036), according to per manufacturer's protocol. qPCR was performed by using SYBR Select Master Mix (Invitrogen, 4472908), with specific gene primers, in StepOnePlus PCR System (Thermo). Each sample was run in triplicate, and target genes expression levels were normalized to β-actin and analyzed by using ∆∆Ct method.
6
Quantitative Gene Expression Analysis
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RNA isolation was performed using the RNeasy Fibrous Tissue Kit (74704, Qiagen) following the manufacturer's instructions. RNA Integrity Number (RIN) was determined in an Agilent 2100 Bioanalyzer (Agilent Technologies). All samples included in the present study showed a RIN score > 7.0. RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). cDNA was obtained from 2 μg of total RNA using the M-MVL reverse transcriptase and oligo-dT12-18 primers according to the manufacturer's instructions (28025013, Thermo Fisher Scientific). 20 ng of cDNA was amplified using the StepOnePlus PCR system (Thermo Fisher Scientific) and the Power SYBR Green PCR Master Mix (4367659, Thermo Fisher Scientific) following the manufacturer's protocol. Table 1 describes the oligonucleotides used in the present study. Negative controls with non-retrotranscribed RNA and without cDNA were included in all the experiments. Relative quantification of gene expression was performed by the ΔΔCt method (relative to the average ΔCT values of the control group), in which 18S ribosomal RNA was used as the endogenous reference gene. All PCR reactions generated a single product of the expected size, as evidenced by melting curve analysis and agarose gel electrophoresis, respectively.
7
Quantitative Real-Time PCR Analysis
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RNA was extracted from hKs, hFs, hiPSCs and hiPSC-bKs using PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA, USA), following the kit’s instructions. RNA concentrations and integrity were measured using Nanodrop 2000 (Thermo Fisher Scientific, San Diego, CA, USA). 500 ng RNA was used for cDNA synthesis using High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA) in a total volume of 20 µL. Reactions were incubated at 37°C for 1h and 95°C for 5 min. cDNA was then diluted 1:10 and 2.5 µL of each diluted cDNA sample was added to 2.5 µL of DNAse-free H2O and 5 µL of TaqMan® Fast Universal PCR Master Mix, making a total volume of 10 µL per reaction in pre-designed Custom Taqman® Array Fast Plates containing previously validated Taqman® assays (Table S2 ) (all from Applied Biosystems, Foster City, CA, USA). Samples were run in a Step One Plus PCR System (Thermo Fisher Scientific, San Diego, CA, USA) in duplicates or triplicates with the following cycle conditions: 95°C for 20s, followed by 40 cycles of 95°C for 1s and 60°C for 20s. Expression levels were determined using the ΔΔCt method and 18S RNA and GAPDH were used independently as reference genes. A minimum of two independent biological replicates were analysed in each experiment.
8
Quantitative Analysis of ApoE and IL-13 in BAL Cells
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Total RNA was extracted from frozen BAL cell pellets using miRNeasy Micro kit (217084, Qiagen, Germantown, MD, USA), according to manufacturer’s protocol. Mouse-specific primers were obtained from Qiagen, for ApoE (PPM04128B) and IL-13 (PPM03021B). GAPDH (PPM02946E) was used as a housekeeping gene. Quantitative-PCR was performed on complementary DNA synthesized using the RT2 First Strand Kit (330404, Qiagen, Germantown, MD, USA) and evaluated on the StepOnePlus PCR System (Thermo Fisher Scientific, Waltham, MA, USA) in comparison to GAPDH using the 2−ΔΔCT method [45 (link)].
9
Nitrate and Rhizobium Regulation of M. truncatula Gene Expression
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To analyze nitrate-induced gene expression, M. truncatula wild-type (R108), cra2, nin-2, or nlp1 seedlings were grown on BNM or FP plates supplemented with 50 nM aminoethoxyvinylglycine (Sigma-Aldrich) for 3 days, then transferred to plates with BNM or FP containing 10 mM KCl or KNO3 for another 3 days. To analyze rhizobium-induced gene expression, R108, nin-2, or nlp1 seedlings were grown on BNM or FP agar plates for 3 days, then inoculated with Sm1021 (OD600 ≈ 0.05) and grown for another 3 days. Total RNA was extracted from M. truncatula roots using the TRIpure isolation reagent (Aidlab, Beijing, China) according to the manufacturer's instructions. RNA was quantified using a NanoDrop 2000 instrument (Thermo Fisher, Waltham, MA, USA) and then reverse transcribed into cDNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China). Gene transcript levels were analyzed by real-time PCR using TB Green Premix Ex Taq (Takara, Dalian, China) with the StepOnePlus PCR system (Thermo Fisher, Waltham, MA, USA). Relative transcript levels were normalized against that of the reference gene (MtUBQ10). Statistical significance based on three biological replicates was calculated using the 2−ΔΔCt method. The primers used for qRT-PCR are listed in Supplemental Table 1 .
10
Quantitative RT-PCR Protocol for Gene Expression
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Total RNA was extracted with TRIzol reagent. Spectrophotometry (TIANGEN, OSE-260, Beijing, China) was used to detect the concentration and purity of RNA. A quantity of 1 μg total RNA was used as a template for reverse transcription PCR to synthesize complementary DNA (cDNA). Quantitative PCR (qPCR) was performed on a Step One plus PCR system (Thermo Fisher Scientific, Waltham, MA, USA) with SYBR green Premix Ex Taq (TakaRa, Kyoto, Japan). The sequences of primers are shown in Supplementary Table S1 .
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