For qRT-PCR analysis, cells were lysed in RTL buffer (Qiagen) supplemented with 1% BME prior to total RNA column purification (Qiagen RNEasy Mini Kit). CDNA generation and real-time PCR reactions were performed using the SuperScript III Platinum One-Step Quantitative RT-PCR kit with Rox by Invitrogen (Carlsbad, California). For MAGE-A3 transcript validation in CRISPR cell lines, Pam 212, Guide 520 and Guide 521 cells were lysed in RTL buffer (Qiagen) supplemented with 1% BME prior to total RNA column purification (Qiagen RNEasy Mini Kit) and DNase treatment (Ambion Turbo DNA Free kit) according to the manufacturers’ instructions. DNase-free RNA was reverse transcribed using SuperScript III (Invitrogen), and cDNA was analyzed by qPCR using Taqman Gene expression assay with primers specific for murine MAGE-A3, MAGE-A4, Cyclin D1, and GAPDH (Table 2 ). RT-PCR was performed using the Applied Biosystems Step-one Real Time PCR system (Foster City, CA).
Rtl buffer
Manufactured by Qiagen
Sourced in Germany, United Kingdom
RTL buffer is a lysis buffer used for the extraction and purification of RNA from biological samples. It is a key component in Qiagen's RNA isolation and purification kits, designed to provide efficient lysis and stabilization of RNA.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (11 mentions)
Rneasy micro kit, by Qiagen (3 mentions)
Facsaria 2 cell sorter, by BD (3 mentions)
Cell strainer cap, by BD (3 mentions)
Rnase free dnase set, by Qiagen (3 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rneasy midi kit, by Qiagen (2 mentions)
Rnasin plus rnase inhibitor, by Promega (2 mentions)
Ultrapure rnase free water, by Qiagen (2 mentions)
Superscript 3 platinum one step quantitative rt pcr kit, by Thermo Fisher Scientific (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
L cysteine, by Worthington (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Worthington (1 mentions)
Steel bead, by Qiagen (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Collection tubes, by Sarstedt (1 mentions)
39 protocols using rtl buffer
1
qRT-PCR Analysis of MAGE-A3 in CRISPR Cell Lines
2
IPEC-J2 Cell Lysis and RNA Extraction
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Differentiated IPEC-J2 cells were lysed at the recovery phase in RTL buffer (Qiagen) scraped and ruptured for 30 s with a tissue rupture probe on ice to generate a homogenous mixture. RNA was isolated following the RNeasy Micro Kit (Qiagen, Germany) procedure as detailed in the user manual. RNA quantity integrity and quality determined by Qubit 4 Fluorometer (Invitrogen) with IQ values above 6.5 utilised and RNA concentrations ˃1.8 µg were used for both Novogene RNA sequencing and qPCR cDNA synthesis (SuperScript®-III).
3
Isolation and Sorting of Cochlear Cells
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For supporting cell sorting, explants were washed in ice cold Ca2+, Mg2+-free -PBS and incubated at 37°C in EBSS solution containing papain (20U/ml), 1mM L-cysteine and 0.5mM EDTA (Worthington, Lakewood, NJ) for 8 minutes (P0 cultured explants) or 10 minutes (all other organs). The papain solution was removed and inactivated by gentle washes in ice cold PBS containing 2% FBS. The cells were dissociated by gently pipetting on ice and filtered with a cell strainer cap (BD Biosciences). The dissociated and filtered cells were sorted in a FACSAriaII cell sorter (BD Biosciences) at 4°C in PBS containing 2% FBS, using a 130μm nozzle. The cells were collected on the basis of their fluorescence gating in DMEM 5% FBS, spun down, lysed in RTL buffer (Qiagen) and stored at -80°C. For each RNAseq library, approximately 50,000 sorted cells were used from the freshly dissected cochleas, and 10,000 sorted cells were used from the cultured explants. The identity of sorted cells was confirmed using epifluorescence and qRT-PCR for hair cell and supporting cell markers. Duplicates samples were collected for each condition.
4
Quantification of Viral Loads in Tissues
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Viral quantification was conducted by qRT-PCR and by Focus Forming Assay (FFA) on serum, spleen, kidney, liver, sciatic nerve, and brain. Sera were collected after centrifugation (15,900 g for 15 min at 4 °C) of blood harvested by cardiac puncture into collection tubes (Sarstedt, #41.1500.005). Following mouse perfusion with PBS, tissues were harvested and stored either in tubes containing RNA Later (Invitrogen) for qRT-PCR or in pre-weighed tubes containing MEMα medium and steel beads (Qiagen, #69989) for FFA. Tissues were then homogenized in RTL buffer (Qiagen) + 1% beta mercaptoethanol for qRT-PCR and MEMα medium for FFA followed by clarification (centrifugation at 2,000 × g for 5 min).
5
Anticoccidial Effects on E. tenella Sporozoites
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Sporozoites (1 × 106 per replicate) of E. tenella Wis strain were pre-incubated for 1 h at 41°C, 5% CO2 with a selection of anticoccidial compounds [amprolium (AMP), robenidine (ROB), and salinomycin (SAL)] or with cytochalasin D (CYT); all compounds were suspended to a final concentration of 5 μg/ml in PBS just before use, made from stock concentrations of 10 mg/ml in dimethyl sulfoxide (DMSO). DMSO alone was also included as a control (0.05% final volume). After incubation, sporozoites were washed with PBS, resuspended in DMEM and added to MDBK monolayers (3 wells/time point/condition, technical replicates). After 2, 24, 44, and 52 hpi cells were recovered using 0.35 ml of RTL buffer (Qiagen) for further DNA extraction. This experiment was performed in duplicate for E. tenella Wis (two biological replicates).
6
Embryonic Intestinal Cell Isolation
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Fifty thousand EpCAM+ embryonic intestinal cells, Lgr5-EGFPhigh ISCs or enterocytes were isolated by FACS directly in 300 μl of RTL buffer (Qiagen) supplemented with 1% β-mercaptoethanol and stored at −80°C. For each replicate, RNA was isolated from pools of 20 embryos (E12.5) or one adult gut using RNeasy micro-kit (Qiagen). 50 ng of total RNA was used for cDNA synthesis using Ovation v2.0 kit (NuGEN) according to manufacturer's instructions. After cDNA fragmentation (Covaris), libraries were prepared using NEBNext v2.0 kit (NEB) according to manufacturer's instructions. Three independent RNA extractions, cDNA synthesis, library preparations and sequencing experiments were performed.
7
Equine Tenocyte Microtissue Generation
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In order to generate equine tenocyte microtissues, cells were adjusted to 2 × 105 cells/mL in normal growth medium and transferred to a reagent reservoir. A total of 25 μL cell suspension was then transferred to individual wells of a Terasaki plate (VWR International, Dietikon, Switzerland) using a multistep pipette. When all wells had been filled, lids were mounted and plates inverted in order to allow for gravity-enforced microtissue formation as previously described [27 (link),33 (link)]. After 6 days of culture, microtissue pellets were harvested and either fixed in 4% paraformaldehyde and processed for paraffin embedding, or lysed in RTL Buffer (Qiagen) and processed for gene expression analysis as described above.
8
Isolation and Sorting of Adipocytes and Stromal Vascular Cells
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Epididymal fat pads were excised from male C57BL/6 mice fed HFD, weighed, and rinsed three times in PBS containing low endotoxin BSA and EDTA (1 mm). Tissue suspensions were subjected to centrifugation at 500g for 5 min and then collagenase treated (1 mg/mL) (Sigma-Aldrich, St. Louis, MO) for 30 min at 37°C with shaking. Cell suspensions were passed through a 100-μm filter and centrifuged at 500g for 5 min. Adipocyte fractions were collected into QIAzol (Qiagen, Valencia, CA) for RNA extraction. Stromal vascular cell fraction pellets were then incubated with erythrocyte lysis buffer (eBioscience, San Diego, CA) for 5 min before centrifugation (300g for 5 min) and resuspended in fluorescence-activated cell sorting buffer. Stromal vascular cell fractions were incubated with Fc Block (BD Biosciences, San Jose, CA) for 20 min at 4°C before staining with fluorescently labeled primary antibodies. The antibodies used were as follows: anti-mouse CD45 Alexa Fluor 700 and anti-mouse F4/80 antigen PerCP-Cyanine5.5. Cells were sorted using a MoFlo sorter (Beckman Coulter, Brea, CA) into RTL buffer (Qiagen).
9
RNA-seq of Arpp21-WT and Arpp21-KO Thymocytes
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DN2 and DN3 thymocytes from Arpp21-WT and Arpp21-KO mice were sorted to >97% purity and stored in RTL buffer (Qiagen) supplemented with fresh β-mercaptoethanol. RNA isolation, library preparation and sequencing were performed by Admera Health (https://www.admerahealth.com/ ). RNA quality was controlled using the Bioanalyzer 2100 Eukaryote Total RNA Nano (Agilent Technologies, CA, USA) and quantity was estimated using Qubit RNA HS assay (ThermoFisher). Low-input RNA poly-A selection libraries were prepared using the SmartSeq V4 with Nextera kit. Samples were sequenced on Illumina 2 × 150, with 20 million reads in each direction. Trimmed RNA-seq reads were pseudo-aligned to the ENSEMBL GRCm38, Ensembl annotation version 101 transcriptome using kallisto, version 0.46. The data have been submitted to the Gene Expression Omnibus under the accession number GSE198798 .
10
Murine Kidney Cell Response to LPS
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Murine kidney single cell suspensions were stimulated in complete RPMI with 100 ng/mL LPS (Sigma-Aldrich) for 2 hrs at 37 °C and 5% CO2. After two washes with ice-cold PBS, cells were either stained for sorting (RNA-seq), or re-suspended in 350 μL of RTL Buffer (Qiagen, Manchester, UK) and processed with QIAshredder homogenizer (Qiagen, Manchester, UK). The follow-through was immediately frozen and stored at −80 °C for not more than two weeks before further processing.
RNA was extracted from thawed samples using Ambion RNA PureLink Kit (Life Technologies, Paisley, UK) and RNA yields analyzed by NanoDrop spectrophotometer (Thermo-Scientific, Loughborough, UK). Complementary DNA (cDNA) was prepared by using High Capacity RNA-to-cDNA™ Kit (Life Technologies, Paisley, UK) and BioRad PCR machine (BioRad, Hemel Hempstead, UK).
RNA was extracted from thawed samples using Ambion RNA PureLink Kit (Life Technologies, Paisley, UK) and RNA yields analyzed by NanoDrop spectrophotometer (Thermo-Scientific, Loughborough, UK). Complementary DNA (cDNA) was prepared by using High Capacity RNA-to-cDNA™ Kit (Life Technologies, Paisley, UK) and BioRad PCR machine (BioRad, Hemel Hempstead, UK).
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