RNA was isolated using the QiaShredder and RNeasy mini kits (Qiagen), treated with RNase-Free DNase 1 (Qiagen), and 0.5 µg was reverse transcribed using the Superscript III Reverse Transcriptase kit (Life Technologies). For the quantitative PCR (qPCR) studies, primer sequences were previously reported as follows: endogenous NANOG, SOX2, OCT4, LIN28A and CHM [13 (link)]; PAX6, ZO1, MERTK, TYR, RLBP1, RDH5 and BEST1 [19 (link)]. RNA from wild type iPSCs [30 (link)] or iPSC-derived RPE [19 (link)] was used as a positive control, and from fibroblasts as a negative control. Reactions were performed in triplicate using the LightCycler® 480 SYBR Green I Master mix on a LightCycler® 480 II thermal cycler (Roche) and analyzed as described [19 (link)]. Quantification was performed using the ΔΔCt method and expression levels were normalized to GAPDH expression.
Qia shredder and rneasy mini kits
Manufactured by Qiagen
Sourced in United States
The QIA-Shredder is a sample disruption and homogenization tool designed to efficiently lyse and homogenize a variety of sample types, including animal and plant tissues, as well as cultured cells, prior to RNA or DNA extraction. The RNeasy mini-kits are a series of kits that provide a simple and reliable method for the isolation and purification of high-quality total RNA from a wide range of sample sources.
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (10 mentions)
Ssoadvanced sybr green supermix, by Bio-Rad (5 mentions)
Iscript cdna synthesis kit, by Bio-Rad (5 mentions)
Superscript 3 reverse transcriptase kit, by Thermo Fisher Scientific (4 mentions)
Rnase free dnase, by Qiagen (3 mentions)
Lightcycler 480 sybr green 1 master mix, by Roche (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480 2, by Roche (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Rnase free dnase 1, by Qiagen (1 mentions)
High capacity cdna kit, by Thermo Fisher Scientific (1 mentions)
Sensifast sybr no rox, by Meridian Bioscience (1 mentions)
Nucleospin rna plus xs kit, by Macherey-Nagel (1 mentions)
Minilys homogenizer, by Bertin Technologies (1 mentions)
Rlt lysis buffer, by Qiagen (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Human ht 12 v4 beadarrays, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
15 protocols using qia shredder and rneasy mini kits
1
Quantitative PCR of iPSC-derived RPE
2
HBV DNA and RNA Isolation, Quantification, and Analysis
3
iPSC-Derived USH2A Expression Analysis
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For the USH2A expression studies, RNA was extracted from iPSCs using the QIAShredder and RNeasy Mini Kits (QIAGEN) and treated with RNase-Free DNase (QIAGEN), and 500 ng was reverse transcribed using the Superscript III Reverse Transcriptase Kit (Thermo Fisher Scientific). qPCR amplification was performed using a 1/10 dilution of cDNA per reaction. Two individual clones per iPSC line were assayed in triplicate and the results averaged; three individual experiments were performed. For the iPSC-derived retinal organoids, RNA was extracted from either individual organoids or pools of 2 organoids using the NucleoSpin RNA Plus XS Kit (Macherey Nagel), and 200 ng was reverse transcribed. qPCR amplifications were performed using a 1/20 dilution of cDNA per reaction in triplicate. For all experiments, reactions were performed with the SensiFAST SYBR No-ROX (Meridian Bioscience) mix on a Roche LightCycler 480 II. Results were normalized to GAPDH expression levels.
4
Fluoxetine Modulates CYP Expression in Worms
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The effects of 0,
0.5, and 20 mg/L fluoxetine on CYP gene expression over time were
analyzed with quantitative real-time PCR (qRT-PCR). 1500 worms were
collected in eppendorf tubes with 300 μL of RLT lysis buffer
(Qiagen, Hilden, Germany) and homogenized using a Minilys homogenizer
(Bertin Technologies) at middle speed four times for 20 s and kept
on ice in between. Total RNA was isolated with the QIAshredder and
RNeasy mini kits (Qiagen) according to the manufacturer’s protocol.
The QuantiTect reverse transcription kit (Qiagen) was used for cDNA
generation. RT-qPCR was performed on a Biorad CFX Opus 384 System
using an iQ SYBR Green Supermix (Biorad) for amplification. The gene
of interest was cyp35-a2, and cdc-42 was used as a housekeeping gene. Primers were generated by Biolegio
(Nijmegen, The Netherlands); further details on the RT-qPCR and primers
can be found inSI A7 . Three biological
replicates were used for each treatment.
0.5, and 20 mg/L fluoxetine on CYP gene expression over time were
analyzed with quantitative real-time PCR (qRT-PCR). 1500 worms were
collected in eppendorf tubes with 300 μL of RLT lysis buffer
(Qiagen, Hilden, Germany) and homogenized using a Minilys homogenizer
(Bertin Technologies) at middle speed four times for 20 s and kept
on ice in between. Total RNA was isolated with the QIAshredder and
RNeasy mini kits (Qiagen) according to the manufacturer’s protocol.
The QuantiTect reverse transcription kit (Qiagen) was used for cDNA
generation. RT-qPCR was performed on a Biorad CFX Opus 384 System
using an iQ SYBR Green Supermix (Biorad) for amplification. The gene
of interest was cyp35-a2, and cdc-42 was used as a housekeeping gene. Primers were generated by Biolegio
(Nijmegen, The Netherlands); further details on the RT-qPCR and primers
can be found in
replicates were used for each treatment.
5
Gene Expression Analysis of Hydrogel-Cultured Cells
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Total RNA was extracted from cell-encapsulated hydrogel constructs or cell spheroids cultured in hydrogel-free medium using QIA-Shredder and RNeasy mini-kits (QIAgen) according to the manufactures’ instructions. Total RNA was synthesized into first strand cDNA in a 20 μL reaction using iScript cDNA synthesis kit (BioRad Laboratories). Real-time PCR analysis was performed in a StepOnePlus™ Real-Time PCR System (Thermo Scientific) using SsoAdvanced SYBR Green Supermix (Bio-Rad). cDNA samples were analyzed for the gene of interest and for the housekeeping gene 18S rRNA. The level of expression of each target gene was calculated using comparative Ct (2–ΔΔCt) method. All primers used in this study are listed in Supplementary Table 1 .
6
Total RNA Extraction and Real-Time PCR Analysis
7
Gene Expression Profiling of Cell Lines
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Gene expression analysis of three biological replicates of each cell line was performed using HumanHT-12 v4 bead arrays from Illumina (San Diego, CA, USA) with ∼48 000 probes. Total RNA was extracted with the QiaShredder and RNeasy Mini Kits (Qiagen, Oslo, Norway) following the manufacturer's instructions. Quality control of RNA was performed with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA amplification, cRNA synthesis, hybridisation, scanning and quantile normalisation were performed as previously described (Halle et al, 2011 (link)). Log2-transformed data were used in further analysis. The data have been deposited to the Gene Expression Omnibus (GEO) repository (GSE80657). Direct HIF1 and TP53 target genes were identified in the data sets by using a list of 276 HIF1 and 129 TP53 targets (Riley et al, 2008 (link); Mole et al, 2009 (link); Xia et al, 2009 (link)). Target genes of mutant TP53 may differ from those depicted in the latter list, but no alternative list was available.
8
Quantitative PCR Analysis of TWIST1 Targets
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All qPCR reactions used an 8-μL mixture of the Applied Biosystems SYBR Green PCR master mix, 0.2 μM forward and reverse primers, and water added to 2 μL of DNA sample. PCR and analysis were done with the Applied Biosystems Fast 7500 system. For validation of ChIP-seq data, primers were designed to flank regions with and without TWIST1-binding sites based on the ChIP-seq analysis. ChIP chromatin from noninduced and 4-d induced HMLE-Twist1ER cells were used, and mRNA levels were compared between the two samples to detect the level of enrichment.
For gene expression qPCR, mRNA was harvested from 70% confluent 60-mm plates using the QIAshredder and RNeasy minikits (Qiagen). Two micrograms of RNA was reverse-transcribed using the high-capacity cDNA reverse transcription kit (Life Technology). Primers used for PCR are listed in the Supplemental Material. All qPCR analyses were performed in triplicates and repeated in three biological replicates that showed consistent results.
For gene expression qPCR, mRNA was harvested from 70% confluent 60-mm plates using the QIAshredder and RNeasy minikits (Qiagen). Two micrograms of RNA was reverse-transcribed using the high-capacity cDNA reverse transcription kit (Life Technology). Primers used for PCR are listed in the Supplemental Material. All qPCR analyses were performed in triplicates and repeated in three biological replicates that showed consistent results.
9
Gene Expression Analysis of Hydrogel Cultures
10
Bone Collagen Gene Expression Analysis
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