RNA was extracted using the RNeasy RNA Micro Kit (Qiagen) per the manufacturer's instructions and quantified using Qubit (ThermoFisher Scientific). One microgram of RNA was then reverse transcribed using SuperScript™ IV Reverse Transcriptase (ThermoFisher, 18090200), dNTP (ThermoFisher, 18427013), RNaseOUT™ Recombinant Ribonuclease Inhibitor (ThermoFisher, 10777019), Oligo(dT)20 Primer (ThermoFisher, 18418020). Quantitative PCR was performed using Kapa SYBR Fast Master mix (Sigma-Aldrich, KK4622) and the primers used were hCSF2RB fwd: 5′ AACGGGATCTGGAGCGAGTG 3′, hCSF2RB rev: 5′ AGATCACGATGAGGGCCAGC 3′, hGAPDH fwd: CTTTTGCGTCGCCAGCCGAG, hGAPDH rev: CCAGGCGCCCAATACGACCA. Each mRNA level was measured as a fluorescent signal normalized based on the signal for β-actin. Relative quantification was determined by the ΔΔCt method and normalized according to β-actin.
Rneasy rna micro kit
Manufactured by Qiagen
Sourced in Germany
The RNeasy RNA micro kit is a laboratory equipment product designed for the purification of total RNA from small tissue samples or cell samples. The kit utilizes a silica-membrane-based technology to isolate and purify RNA, which can then be used for various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Powerup syber green master mix, by Thermo Fisher Scientific (2 mentions)
Iscript reverse transcription supermix, by Bio-Rad (2 mentions)
High capacity rna to cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Ananodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Sso advanced preamp supermix kit, by Bio-Rad (2 mentions)
Rnaseout recombinant ribonuclease inhibitor, by Thermo Fisher Scientific (1 mentions)
Oligo dt 20 primer, by Thermo Fisher Scientific (1 mentions)
Kapa sybr fast master mix, by Merck Group (1 mentions)
Superscript 4 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
Superscript vilo master mix, by Thermo Fisher Scientific (1 mentions)
Qscript cdna supermix, by Avantor (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Experion system, by Bio-Rad (1 mentions)
Iq supermix, by Bio-Rad (1 mentions)
Rest2009 program, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Mirneasy micro kit, by Qiagen (1 mentions)
Taqman array human microrna a card, by Thermo Fisher Scientific (1 mentions)
6 protocols using rneasy rna micro kit
1
Quantitative RT-PCR for Gene Expression
2
RNA Extraction and qPCR Analysis of Cardiac and Macrophage Samples
3
RNA Extraction and qPCR Analysis of Cardiac and Macrophage Samples
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RNA was extracted from the cardiac slices or cultured cells using
the RNeasy RNA micro kit (Qiagen). RNA concentration was measured using a
nanodrop spectrophotometer (ThermoFisher Scientific). For cardiac slices,
cDNA synthesis was performed using the High Capacity RNA to cDNA synthesis
kit (Applied Biosystems). For cultured macrophages, cDNA was synthesized
using the iScript™ Reverse Transcription Supermix (Bio-Rad) and
pre-amplified using the Sso Advanced PreAmp Supermix kit (Bio-Rad).
Quantitative real time PCR reactions were prepared with sequence-specific
primers (IDT) with PowerUP™ Syber Green Master mix (ThermoFisher
Scientific) in a 20 μL volume. Real time PCR was performed using
QuantStudio 3 (ThermoFisher Scientific). mRNA expression was normalized to
β2 Microglobulin (B2M). IL-1β: Forward ATG CAC CTG TAC GAT
CAC TG, Reverse ACA AAG GAC ATG GAG AAC ACC; CCL7: Forward AGA CCA AAC CAG
AAA CCT CC, Reverse AGT ATT AAT CCC AAC TGG CTG AG; IL-10: Forward CGC ATG
TGA ACT CCC TGG, Reverse TAG ATG CCT TTC TCT TGG AGC; TNF: Forward ACT TTG
GAG TGA TCG GCC, Reverse GCT TGA GGG TTT GCT ACA AC; β2M: Forward
TGC TGT CTC CAT GTT TGA TGT ATC T, Reverse TCT CTG CTC CCC ACC TCT AAG.
the RNeasy RNA micro kit (Qiagen). RNA concentration was measured using a
nanodrop spectrophotometer (ThermoFisher Scientific). For cardiac slices,
cDNA synthesis was performed using the High Capacity RNA to cDNA synthesis
kit (Applied Biosystems). For cultured macrophages, cDNA was synthesized
using the iScript™ Reverse Transcription Supermix (Bio-Rad) and
pre-amplified using the Sso Advanced PreAmp Supermix kit (Bio-Rad).
Quantitative real time PCR reactions were prepared with sequence-specific
primers (IDT) with PowerUP™ Syber Green Master mix (ThermoFisher
Scientific) in a 20 μL volume. Real time PCR was performed using
QuantStudio 3 (ThermoFisher Scientific). mRNA expression was normalized to
β2 Microglobulin (B2M). IL-1β: Forward ATG CAC CTG TAC GAT
CAC TG, Reverse ACA AAG GAC ATG GAG AAC ACC; CCL7: Forward AGA CCA AAC CAG
AAA CCT CC, Reverse AGT ATT AAT CCC AAC TGG CTG AG; IL-10: Forward CGC ATG
TGA ACT CCC TGG, Reverse TAG ATG CCT TTC TCT TGG AGC; TNF: Forward ACT TTG
GAG TGA TCG GCC, Reverse GCT TGA GGG TTT GCT ACA AC; β2M: Forward
TGC TGT CTC CAT GTT TGA TGT ATC T, Reverse TCT CTG CTC CCC ACC TCT AAG.
4
Quantifying mRNA Expression in Cells
5
Quantitative PCR Analysis of α-SMA
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Three gels per condition were pooled, solubilized in Trizol (Invitrogen) and RNA isolated using the RNeasy Micro RNA kit with on column DNase I digestion as recommended by the manufacturer (Qiagen, Valencia, CA). RNA concentrations were determined using a BioRad Experion system with standard sensitivity RNA chips and 50 ng of total RNA was used to prepare cDNA using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). PCR reactions were carried out using iQ Supermix (BioRad) with primers specific for rat α -smooth muscle actin (α-SMA) (forward: 5′ GGAGTGATGGTT-GGAATGG 3′; reverse: 5′ ATGATGCCGTGTTCTATCG 3′) and control gene acidic ribosomal phosphoprotein (ARBP) (forward: 5′ TAGAGGGT-GTCCGCAATG 3′; reverse: 5′ GAAGGTGTAGTCAGTC-TCC 3′). Data were analyzed using the fold change method incorporating primer efficiency with the REST2009 program (Qiagen Inc., Valencia, CA).
6
Quantifying gene and miRNA expression in iPSCs
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Total RNA from cultured cells was extracted using an RNeasy Mini Kit or RNeasy Micro RNA Kit (Qiagen, Hilden, Germany) based on the manufacturer's instructions. Total RNA (1 μg) was used for cDNA synthesis using SuperScript BMP4 dose effect on human iPSCs J Kojima et al III (ThermoFisher Scientific). cDNA was used for TaqMan gene expression analysis. The primers used in this study are shown in Supplementary Table S1. For normalization, GAPDH was used as an internal control in the TaqMan array (and individual TaqMan probe assay). Genes that were clearly detected in two experiments were used for the assay.
For miRNA detection, a miRNeasy Micro Kit (Qiagen) was used for extraction of RNA and cDNA synthesis according to the manufacturer's instructions. miRNA was analyzed using a TaqMan Human MicroRNA array card A (ThermoFisher Scientific). U6 was used as an internal control.
For miRNA detection, a miRNeasy Micro Kit (Qiagen) was used for extraction of RNA and cDNA synthesis according to the manufacturer's instructions. miRNA was analyzed using a TaqMan Human MicroRNA array card A (ThermoFisher Scientific). U6 was used as an internal control.
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