For qRT-PCR, total RNA from sorted MEC subsets was prepared by the RNeasy kit and cDNA was generated with iScript (Bio-Rad) according to the manufacture’s protocol. PCR was performed using FastStart SYBR Green Master (Roche).
Rneasy kit
Manufactured by Bio-Rad
Sourced in United States, Germany
The RNeasy kit is a laboratory product designed for the purification of total RNA from various sample types. It utilizes a silica-membrane-based technology to efficiently capture and isolate RNA molecules. The core function of the RNeasy kit is to provide a reliable and reproducible method for the extraction and purification of high-quality RNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (12 mentions)
Cfx96 thermal cycler, by Bio-Rad (3 mentions)
Itaq universal sybr green supermix, by Bio-Rad (2 mentions)
Sybr green assay, by Bio-Rad (2 mentions)
Miniopticon system, by Bio-Rad (2 mentions)
Faststart sybr green master, by Roche (1 mentions)
Iscript, by Bio-Rad (1 mentions)
Iqtm sybr green supermix, by Bio-Rad (1 mentions)
Iq5 thermal cycler, by Bio-Rad (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Lightcycler 96 thermal cycler, by Roche (1 mentions)
Viia real time pcr system, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Bio-Rad (1 mentions)
Sybr green mix, by Thermo Fisher Scientific (1 mentions)
Iscript select cdna synthesis kit, by Bio-Rad (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Iscript kit, by Bio-Rad (1 mentions)
Rt2 qpcr primer assay, by Qiagen (1 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
Mastercycler, by Bio-Rad (1 mentions)
17 protocols using rneasy kit
1
Quantitative RT-PCR of Mammary Epithelial Cells
2
Quantifying BMP4 Expression in HUVECs and Mouse Tissues
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Total RNA from cultured HUVECs or previously homogenized mouse tissues was isolated and purified using the SpeedTools kit (Biotools), or the RNeasy kit (170-8891; iScript cDNA Synthesis kit; BioRad). One μg of total RNA from each sample was retrotranscribed into cDNA with the iScript cDNA Synthesis kit (BioRad) in a final volume of 20 μL, following the manufacturer’s instructions. The resulting cDNA was used as a template for subsequent quantitative real-time PCR. For qRT-PCR assays of human BMP4, specific oligonucleotides labeled with FAM (Hs03676628_s1; TaqMan Gene Expression Assays, Applied Biosystems), and Roche’s FastStart Essential DNA Probes Master Mix containing Taq DNA Polymerase (Life Science). Amplification experiments were performed with the iQ5 thermal cycler (Bio-Rad). The qRT-PCR of murine Bmp4 was carried out using the iQTM SYBR® Green Supermix (170-8880; BioRad), and mouse Bmp4 specific oligonucleotides (Forward, CGTTACCTCAAGGGAGTGGA; Reverse, ATGCTTGGGACTACGTTTGG). DNA amplification was performed with the Roche LightCycler 96 thermal cycler, using human or murine 18S ribosomal RNA as an internal control. Samples were analyzed in triplicate, and each experiment was repeated at least three times. Results were normalized with respect to the expression levels of the 18S ribosomal RNA by the 2−ΔΔCt method.
3
Quantitative Real-Time PCR for Gene Expression
4
Quantitative Analysis of BMP6 and Hepcidin
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Total cellular RNA was extracted from cell monolayers with a RNeasy Kit following manufacturer’s instructions, and then cDNA was synthesized by iScript select cDNA synthesis kit (Bio-Rad; Hercules, CA). Real-time RT-PCR was performed with the iTaq Universal SYBR Green Supermix (Bio-Rad) on 7500 Fast Real-time PCR System (Applied Biosystems) following the manufacturer’s instruction. Primer sequences are as follows:
BMP6 (human)
forward 5′-CGTGAAGGCAATGCTCACCT-3′,
reverse 5′- CCTGTGGCGTGGTATGCTGT-3′,
BMP6 (murine)
forward 5′- AAGACCCGGTGGTGGCTCTA-3′,
reverse 5′-CTGTGTGAGCTGCCCTTGCT-3′,
hepcidin (human)
forward 5′-GACGGGACAACTTGCAGAGC-3′,
reverse 5′-GCCTCTGGAACATGGGCA-3′,
hepcidin (murine)
forward 5′-AACAGATACCACACTGGGAA-3′,
reverse 5′-CCTATCTCCATCAACAGATG-3′,
β-Actin (human)
forward 5′-ACGGTGAAGGTGACAGCAGTCG-3′,
reverse 5′-AATGTGCAATCAAAGTCCTCGGC-3′,
β-Actin (murine)
forward 5′-CCGTGAAAAGATGACCCAGA-3′,
reverse 5′-AGGCATACAGGGACAGCACA-3′
BMP6 (human)
forward 5′-CGTGAAGGCAATGCTCACCT-3′,
reverse 5′- CCTGTGGCGTGGTATGCTGT-3′,
BMP6 (murine)
forward 5′- AAGACCCGGTGGTGGCTCTA-3′,
reverse 5′-CTGTGTGAGCTGCCCTTGCT-3′,
hepcidin (human)
forward 5′-GACGGGACAACTTGCAGAGC-3′,
reverse 5′-GCCTCTGGAACATGGGCA-3′,
hepcidin (murine)
forward 5′-AACAGATACCACACTGGGAA-3′,
reverse 5′-CCTATCTCCATCAACAGATG-3′,
β-Actin (human)
forward 5′-ACGGTGAAGGTGACAGCAGTCG-3′,
reverse 5′-AATGTGCAATCAAAGTCCTCGGC-3′,
β-Actin (murine)
forward 5′-CCGTGAAAAGATGACCCAGA-3′,
reverse 5′-AGGCATACAGGGACAGCACA-3′
5
RNA Extraction from Frozen Plants
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Frozen pollen or leaf samples were ground in liquid nitrogen with a mortar and pestle and RNA was extracted using a RNeasy kit (Bio-Rad catalog number 74904: Bio-Rad; Hercules, California, USA) following manufacturer’s instructions. RNAs were DNaseI treated via an on-column digestion with the Plant RNeasy kit. RNA quality was assessed with an Agilent BioAnalyzer and only high quality samples were used.
6
Comprehensive Liver Gene Expression Analysis
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Assessment of gene expression at the mRNA level in liver tissue or cultured cells was performed by real time quantitative PCR (RT-qPCR) for Ghr, GHS-R1, CK19, PCNA, desmin, αSMA, Col1A1, MMP2, TIMP1, integrin β6, integrin αv, FN1, TGFβ, PDGFα, CTGF, CCL2, IL-1β, IL-6. Fold changes in gene expression were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Total RNA was isolated by using RNeasy kit, followed by cDNA synthesis with iScript kit from Bio-Rad Life Sciences (Hercules, CA), and RT-qPCR using iTaq Universal SYBR-Green Supermix from the same company. RT2 qPCR Primer Assays were purchased from Qiagen (Frederik, MD). A list of all primers used, is presented in supplemental Table 1 . AriaMx Real-Time PCR system thermal cycler from Agilent Technologies (Santa Clara, CA) was used for running RT-qPCR. The data was analyzed as previously described65 (link).
7
RT-PCR Characterization of ZEST Cells
8
qRT-PCR Analysis of ZEST Cell Gene Expression
9
Quantitative gene expression analysis
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RNA was extracted from cells using the Qiagen RNeasy kit, and Bio-Rad’s iScript cDNA Synthesis kit generated cDNA. Quantitative real-time PCR was performed using the Bio-Rad CFX96 thermal cycler and primers are listed in Supplemental Table 1 . The use of GAPDH as a housekeeping transcript was validated by comparison to a panel of commonly used reference cDNAs.
10
Quantitative gene expression analysis
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