Total RNA was isolated from skin tissues with Trizol reagent (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The quality and quantity of the isolated RNA were determined spectrophotometrically and by agarose gel electrophoresis. Five μg (5 μg) of total RNA was then reverse-transcribed into cDNA using Superscript first-strand synthesis kit or RT-PCR (Life Technologies). Subsequently, quantitative real-time PCR was performed on a StepOne PCR System in MicroAmp® Fast Optical 48-well reaction plates (both from Applied Biosystems, Thermo Fisher Scientific) using the KAPA SYBR®FAST qPCR Kit (Kapa Biosystems, Wilmington, DE, USA) under the following conditions: 95 °C for 3 min followed by 40 cycles of 95 °C for normalization. Each reaction was performed in triplicate and each experiment included two non-template controls. The sequences of MMP1, MMP3, MMP7, MMP9, and β-actin primers are shown in Table 2 . Primer specificity was verified by melting curve analysis. For relative quantification of the transcripts, the formula RQ = 2−ΔΔCt was used.
Stepone pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, Belgium
The StepOne PCR system is a real-time PCR instrument designed for reliable and efficient DNA amplification and analysis. It features a compact, space-saving design and provides accurate and reproducible results for a variety of real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (13 mentions)
Rneasy mini kit, by Qiagen (10 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Stepone software v2, by Thermo Fisher Scientific (5 mentions)
Nanodrop spectroscopy, by Thermo Fisher Scientific (4 mentions)
Qscript cdna synthesis kit, by Quanta Biosciences (4 mentions)
Qscript cdna synthesis kit, by Avantor (4 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions)
Kapa sybr fast qpcr kit, by Roche (3 mentions)
Microamp fast optical 48 well reaction plate, by Thermo Fisher Scientific (3 mentions)
Taqman probe, by Thermo Fisher Scientific (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (3 mentions)
Superscript 3, by Thermo Fisher Scientific (3 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (3 mentions)
Kapa sybr green, by Merck Group (3 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Tri reagent, by Merck Group (2 mentions)
Rnalater, by Qiagen (2 mentions)
Sybr green master mix, by Bio-Rad (2 mentions)
Superscript first strand synthesis system for rt pcr, by Thermo Fisher Scientific (2 mentions)
70 protocols using stepone pcr system
1
Quantitative RT-PCR Analysis of MMPs
2
Quantitative Gene/miRNA Expression in Mouse Lungs
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RNA was extracted from frozen lungs of 3-week-old mice using TRIzol reagent (Life Technologies) and quantified by Nanodrop spectroscopy (Thermo Scientific). cDNA was generated from 1 μg RNA by reverse transcription using qScript cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD). Real-time quantitative polymerase chain reaction was performed on a StepOne PCR system (Applied Biosystems, Foster City, CA) using TaqMan probes (Life Technologies) for GSNOR (Mm00475804_g1) compared with 25% diluted β-actin control (Thermo Scientific) with PerfeCTa qPCR FastMix, UNG, ROX (Quanta Biosciences, Gaithersburg, MD). For microRNA qRT-PCR, RNA was similarly extracted as in the miR microarray studies. cDNA was generated using TaqMan primer-specific assays and MicroRNA Reverse Transcription kit, and real-time quantitative polymerase chain reaction was performed using TaqMan MicroRNA assays for microRNA-342-3p (2260, Thermo Scientific) compared with snRNA-U6 control (001973, Thermo Scientific) with TaqMan Universal Master Mix, No AmpErase UNG (Life Technologies). Fold changes are reported utilizing 2^-ddCT method and StepOne software v2.3 (Applied Biosystems).
3
Comprehensive Steroidogenic Gene Expression
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Total RNA was extracted using the mini kit for RNeasy AT (Qiagen, Germany). A
spectrophotometer (BioRad, Stanford, USA) was used for evaluation of the quality of the
extracted RNA in terms of the A260/280 ratio. Then, for cDNA synthesis on RNA samples,
Prime Script RT Reagent Kit (Takara, Japan) was used and all experiments included negative
controls (without cDNA) and RT controls. Gel electrophoresis were used for analysis of PCR
products. Quantitative reverse-transcription polymerase chain reaction (qRTPCR) was
performed on the Step-One PCR system (Applied Biosystems, USA), mRNA quantification was
conducted and each reaction was run in duplicate. The NCBI primer Blast and Perl primer
Software (version1.1.21) were used for primer design of all steroids target genes.
Ultimately, GAPDH as the housekeeping gene was used and messenger RNA
expression levels of all genes were analyzed by Qrt-pcr (2−ΔΔct). We analyzed
expression level of 14 genes (7 (link)): steroidogenic acute regulator (STAR),
cytochrome P450 monooxygenase (CYP11A1), 17α-hydroxylase
(CYP17A1), steroid 21-hydroxylase (CYP21),
11β-Hydroxysteroid dehydrogenase (11BHSD1 and 11BHSD2),
aromatase cytochrome P450 (CYP19A1), 3β-hydroxysteroid dehydrogenase
(3BHSD1 and 3BHSD2) and 17 hydroxysteroid
dehydrogenase family (17BHSD types 1, 3 (link), 5 (link), 7 and 12).
spectrophotometer (BioRad, Stanford, USA) was used for evaluation of the quality of the
extracted RNA in terms of the A260/280 ratio. Then, for cDNA synthesis on RNA samples,
Prime Script RT Reagent Kit (Takara, Japan) was used and all experiments included negative
controls (without cDNA) and RT controls. Gel electrophoresis were used for analysis of PCR
products. Quantitative reverse-transcription polymerase chain reaction (qRTPCR) was
performed on the Step-One PCR system (Applied Biosystems, USA), mRNA quantification was
conducted and each reaction was run in duplicate. The NCBI primer Blast and Perl primer
Software (version1.1.21) were used for primer design of all steroids target genes.
Ultimately, GAPDH as the housekeeping gene was used and messenger RNA
expression levels of all genes were analyzed by Qrt-pcr (2−ΔΔct). We analyzed
expression level of 14 genes (7 (link)): steroidogenic acute regulator (STAR),
cytochrome P450 monooxygenase (CYP11A1), 17α-hydroxylase
(CYP17A1), steroid 21-hydroxylase (CYP21),
11β-Hydroxysteroid dehydrogenase (11BHSD1 and 11BHSD2),
aromatase cytochrome P450 (CYP19A1), 3β-hydroxysteroid dehydrogenase
(3BHSD1 and 3BHSD2) and 17 hydroxysteroid
dehydrogenase family (17BHSD types 1, 3 (link), 5 (link), 7 and 12).
4
RNA Extraction and Expression Analysis
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RNA was extracted from samples using the Trizol reagent (Takara, Japan). The Thermo Scientific NanoDrop spectrophotometer was used to detect the purity and concentration of RNA. Then, cDNA was synthesized using the reverse transcriptase kit (Takara, Japan). Quantitative analysis of mRNA expression was conducted with a SYBR premix EX TaqTM kit (Takara, Japan) with StepOne PCR system (Applied Biosystems, USA). The relative quantity of mRNA was expressed as 2-△△CT. Sequences of the primers are listed in Supplementary Table S2 .
5
Colonic Organoid Inflammation Response
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Samples were immediately processed to yield isolated colonic crypts as previously described (Fernando et al, 2017 ). Isolated colonic crypts were embedded in Matrigel® (Corning) and cultured in Intesticulttm Organoid Growth Media. Human organoids were pretreated for 6 h with either FKK5, FKK6, FKK9, or vehicle (DMSO), then stimulated with human recombinant TNF‐alpha in the presence of either treatment for an additional 12 h. Organoids were washed in PBS, disrupted in Tri Reagent® (Sigma, Oakville, ON, CAN), and frozen at −80°C. After thawing, chloroform was added, samples were centrifuged for 15 min, and the resulting aqueous phase was then added to equal volumes of 70% ethanol and further processed using the RNeasy mini kit (Qiagen). cDNA was synthesized using the QuantiTect RT kit (Qiagen) according to the manufacturer's protocol. Resulting cDNA was used as a template for quantitative real‐time PCR using Perfecta SYBR Green FastMix with ROX (QuantaBio). PCR and analysis were performed using a StepOne PCR System (Applied Biosystems). Gene expression was calculated relative to β‐actin expression and expressed as fold change of control. The primers used were as follows: Human®‐Actin (NM_001101), Human CYP3A4 (NM_001202855), and Human ABCB1 (NM_000927).
6
Quantitative RNA Expression Analysis
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Total RNA was extracted from FTECs using the RNeasy Mini Kit (#74104, Qiagen, Germantown, MD, USA). Double-stranded cDNA was synthesized using a reverse transcription kit (#4368813, ThermoFisher Scientific). Real-time quantitative polymerase chain reaction (RT-PCR) was performed by the FastStart Universal Probe Master kit (#04913957001, Roche) with Roche Universal Probe #2 and Probe #30 (#04684982001 and #04687639001, Roche) using a StepOne PCR system (Applied Biosystems, Foster, CA, USA). Relative RNA quantitation was performed using ∆∆CT calculations. Primer sequences are listed in Supplementary Table S2 .
7
RNA Extraction and qRT-PCR Analysis
8
RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated from tissue sections stored in Qiagen RNAlater using the Qiagen RNeasy Mini Kit according to the manufacturer's protocol with on-column DNase digestion using the Qiagen RNase-Free DNase Set. cDNA was generated using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer's protocol. cDNA was used as a template for quantitative real-time PCR using SYBR Green Master Mix (Bio-Rad). PCR and analysis was performed using a StepOne PCR system (Applied Biosystems). Gene expression was calculated relative to that of gapdh.
9
Examining CFTR Expression in G542X Organoids
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To examine Cftr expression, G542X intestinal organoids were passaged into 24-well plates, with 35 µL of MatriGel per well. One well was considered to be a single experiment. The organoids were grown for five days to the point of being significantly budded, and were then treated for 24 h with indicated compounds diluted in 500 µL OGM. The organoids were then lysed using a QiaShredder Cell and Tissue Homogenizer Kit (Qiagen, Germantown, MD, USA, cat. #79654). RNA was harvested using an RNeasy Mini Kit (Qiagen, cat. #74104). 250 ng of RNA was reverse transcribed into cDNA using a QScript cDNA synthesis kit (VWR, Radnor, PA, USA, cat. #101414-098). Cftr expression was examined using a TaqMan expression assay which used primers recognizing exons 17 and 18 on Cftr (Mm00445197; ThermoFisher Scientific,). RTqPCR was performed on a StepOne PCR system (Applied Biosystems) to examine Cftr expression, with ß-actin serving as the endogenous control. The average of each value was expressed as the fold change difference in Cftr expression from DMSO-treated organoids.
10
Notch Pathway Expression in Mouse Retina
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