Non-specific binding of the immunoglobulin to the Fc receptors was blocked by incubating cells 10 min on ice in 0.5% BSA in DPBS (without Ca2+ or Mg2+) with 2 mM EDTA supplemented with TruStain fcX (anti-mouse Cd16/32, BioLegend, #101319, clone 93, 1:100). Cells were stained with anti-CD11B-Alexa647 (clone M1/70, 1:100) and anti-CD45-PE-Cy7 (clone 30-F11, 1:100) for 30 min on ice. Finally, cells were washed with 1 mL DPBS (without Ca2+ or Mg2+) with 0.5% BSA and 2 mM EDTA and were centrifuged at 300 × g for 10 min, resuspended in 0.5% BSA, 2 mM EDTA in DPBS (without Ca2+ or Mg2+) and passed through a 35 mm nylon mesh strainer (BD Falcon). DAPI was added to cells at final concentration of 1 μg/ml. Live cells were sorted using a BD FACSAria II SORP Cell Sorter. Sorted cells were directly lysed in RLT Plus buffer (QIAGEN) and RNA was extracted using the RNeasy Plus RNA isolation kit (QIAGEN) according to manufactures protocol following appendix D.
Rneasy plus rna isolation kit
Manufactured by Qiagen
Sourced in Sweden
The RNeasy Plus RNA isolation kit is a product designed for the purification of high-quality total RNA from various sample types. It utilizes a silica-based membrane technology to efficiently capture and purify RNA, including small RNA species. The kit provides a streamlined workflow for the isolation of RNA, ensuring consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (8 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (8 mentions)
Rlt plus buffer, by Qiagen (3 mentions)
Ssofast evagreen supermix, by Bio-Rad (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Itaq universal sybr green supermix, by Bio-Rad (3 mentions)
Perfecta sybr green fastmix, by Quanta Biosciences (3 mentions)
Facsaria 2 sorp cell sorter, by BD (2 mentions)
35 mm nylon mesh strainer, by BD (2 mentions)
Trustain fcx, by BioLegend (2 mentions)
Cfx connect real time system, by Bio-Rad (2 mentions)
Kicqstart, by Merck Group (2 mentions)
Mouse pan cancer immune oncology kit, by NanoString (2 mentions)
Nsolver analysis software, by NanoString (2 mentions)
Liberase, by Roche (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Qiashredder, by Qiagen (1 mentions)
Qscript cdna supermix, by Quanta Biosciences (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Dna free kit, by Thermo Fisher Scientific (1 mentions)
23 protocols using rneasy plus rna isolation kit
1
Isolation and Characterization of Immune Cells
2
RNA Extraction and cDNA Preparation
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LADR and 2 cells were harvested, and total RNA was extracted from 1 × 106 LAD2 cells using the RNeasy Plus RNA isolation kit (Qiagen, Germantown, MD, USA) as described [10 (link)]. Approximately 1 µg of total RNA was reverse transcribed (RT) using the SuperScript III First-Strand synthesis system (Invitrogen, Grand Island, NY, USA) with random hexamer primers. cDNA and expression arrays comparing LADR with LAD2 gene expression were then prepared using RT-PCR according to manufacturers’ recommendations. Sequencing data was analyzed using Sequencher (Version 4.5, Softgenetics, State College, PA, USA).
3
Quantitative Real-Time PCR Analysis
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Cells were harvested for total RNA isolation using the RNeasy Plus RNA isolation kit (Qiagen). cDNA synthesis was performed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). Quantitative real-time PCR (qRT-PCR) using QuantIT Perfecta Supermix was performed with the CFX96 Real-Time PCR Detection System (Bio-Rad) with the oligonucleotide primers reported in Supplementary Table 2 . The results are expressed as fold-increase mRNA expression of the gene of interest normalized to Gapdh expression by the ΔΔCt method.
4
Isolation and Characterization of Immune Cells
5
Biobot Skeletal Muscle Transcriptome Analysis
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Muscle rings were removed from biobot skeletons after force date collection and snap-frozen (5 min in liquid nitrogen). Samples were then stored at −80 °C and thawed on the day of RT-qPCR experiment. RNA was extracted using the RNeasy Plus RNA isolation kit (Qiagen), following the instructions provided. For quantitative PCR (qPCR), TaqMan fast advanced Master Mix, myogenic health PCR targets, and housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were added to cDNA. PCR was performed on QuantStudio3: 2 min at 50 °C, 2 min at 95 °C, 40 cycles of 1 s at 95 °C and 20 s at 60 °C. Cycle threshold (Ct) values were obtained to calculate changes in expression level using by the 2−ΔΔCt method.32 (link)
6
Quantitative PCR Analysis of Gene Expression
7
Quantification of Repetitive Genomic Sequences
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RNAs were isolated using RNeasy Plus RNA isolation kit (Qiagen) and additionally purified of DNA contamination using DNA-free kit (Thermo Fisher). 2 μg of RNA was reverse-transcribed using High Capacity cDNA Reverse Transcription kit (Applied Biosystems) per manufacturer’s protocol, either with random primers, or with a GAPDH-reverse primer 5′GATGCAGGGATGATGTTCTG-3′ and one of the following: an HSATII primer 5′ATCGAATGGAAATGAAAGGAGTCA-3′ or an HSATIII primer 5′TCCATTCCATTCCTGTACTCGG-3′. cDNAs were diluted 1:2 and 1 μL of diluted cDNA was used per qPCR reaction. Reactions were run in triplicates with iTaq Universal SYBR Green supermix (Bio-Rad) and the following pairs of primers: 5′ACAACTTTGGCATTGAA-3′ and 5′GATGCAGGGATGATGTTCTG-3′ for GAPDH, described in76 (link); 5′TCATCGAATGGAAATGAAAGGAGTCATCATCT-3′ and 5′CGACCATTGGATGATTGCAGTCAA-3′ for HSATII; 5′AATCAACCCGAGTGCAATCGAATGGAATCG-3′ and 5′TCCATTCCATTCCTGTACTCGG-3′ for HSATIII, described in Shen et al.75 (link) Triplicate Ct values for GAPDH were averaged and subtracted from HSAT values to derive ΔCt values. The results were plotted as mean 2–ΔCt values. Significance was determined in paired two-tailed t-tests.
8
Quantitative Real-Time PCR Analysis
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RNA was isolated using the RNeasy Plus RNA isolation kit (Qiagen). cDNA synthesis was performed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). Real-time polymerase chain reaction (PCR) using PerfeCTa SYBR Green FastMix (Quanta Biosciences) was performed with the CFX96 Real-Time PCR Detection System (Bio-Rad). Oligonucleotide primers and PCR conditions are reported in Supplementary Table S1 . Primer specificity was confirmed by agarose gel electrophoresis and melting curve analysis. Reaction efficiencies over the appropriate dynamic range were calculated to ensure linearity of the standard curve (13 (link)). The results are expressed as fold-increase mRNA expression of the gene of interest normalized to Beta Actin expression using the ΔΔCt method. Reported values are the mean and S.E.M. from two independent experiments (n = 2) where technical replicates were averaged for each experiment. Effects were evaluated with multivariate ANOVA and Dunnett’s post hoc test using JMP 10 Pro.
9
Gene Expression Analysis by RT-qPCR
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RNA was isolated using the RNeasy Plus RNA isolation kit (Qiagen). cDNA was synthesized with the SuperScript VILO cDNA Synthesis Kit (Invitrogen). Real-time PCR using PerfeCTa SYBR Green FastMix (Quanta Biosciences) was performed with the CFX96 Real-Time PCR Detection System (Bio-Rad). The results are expressed as fold-increase expression of the gene of interest normalized to GAPDH expression using the ΔΔCt method.
10
RNA Isolation and qRT-PCR Analysis
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Total RNA was isolated using the RNeasy Plus RNA isolation kit (Qiagen), and cDNA synthesis was performed using the SuperScript VILO cDNA synthesis kit (Invitrogen). Real-time PCR using PerfeCTa SYBR Green FastMix was performed on the CFX96 Real-Time PCR Detection System (Bio-Rad) with the oligonucleotide primers shown in Supplementary Table 2 . Primer specificity was confirmed by agarose gel electrophoresis and melting curve analysis. The results are expressed as the fold increase in mRNA expression of the gene of interest normalized to that of GAPDH. The values are reported as the mean ± s.e.m. from three independent experiments performed on different days (n = 3) with technical duplicates that were averaged for each experiment.
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