Fluorescence-activated cell sorted peritoneal B1a, B1b, and B2 B cells were loaded onto a QIAshredder spin column (QIAGEN, Hilden, Germany) and homogenized. For RNA isolation from the lysate, the RNeasy Mini Kit (QIAGEN) was used according to the manufacturer’s instructions. RNA concentration was measured by a Tecan Infinite N200 PRO (Tecan Austria GmbH, Gröding, Austria). Identical amounts of RNA (30 ng) were used for cDNA synthesis with the QuantiTect Reverse Transcription Kit (QIAGEN). Real-time RT-PCR for S1P11–5 receptor expression was performed with the QuantiTect SYBR Green PCR Kit (QIAGEN) on an ABI Prism 7000 Sequence Detection System (SDS) (Applied Biosystems, Foster City, CA, USA). Analysis was done with the ABI Prism 7000 SDS software v1.1 (Applied Biosystems). Semi-quantitative gene expression was calculated according to the 2−ΔCt method normalized to β2 microglobulin. The consistency of the internal control was verified by comparing 2−ΔΔCt values as described by Schmittgen et al. [44 (link)]. Primers were designed using Primer3 software (version 0.4.0, Whitehead Institute for Biomedical Research, Cambridge, MA, USA) and synthetized by BIOTEZ (Berlin, Germany). Primer sequences are listed in Table 1 .
Qiashredder spin column
Manufactured by Qiagen
Sourced in Germany, United States
The QIAshredder spin columns are a laboratory tool used to homogenize and disrupt cells or tissues prior to RNA or DNA extraction. The columns employ a unique design to effectively shear and lyse samples, facilitating the release of nucleic acids for further downstream processing.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (58 mentions)
Rneasy plus mini kit, by Qiagen (13 mentions)
Rlt buffer, by Qiagen (11 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (11 mentions)
Allprep dna rna mini kit, by Qiagen (9 mentions)
Rneasy kit, by Qiagen (8 mentions)
Mirneasy mini kit, by Qiagen (8 mentions)
Rna 6000 nano labchip, by Agilent Technologies (7 mentions)
Qiazol lysis buffer, by Qiagen (7 mentions)
Rlt plus buffer, by Qiagen (6 mentions)
Ds 11 nanodrop spectrophotometer, by DeNovix (6 mentions)
Agilent bioanalyzer, by Agilent Technologies (6 mentions)
Quantitect reverse transcription kit, by Qiagen (5 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Rnalater, by Thermo Fisher Scientific (4 mentions)
Rneasy plus micro kit, by Qiagen (4 mentions)
Genespring software, by Agilent Technologies (4 mentions)
Codelink expression analysis software, by GE Healthcare (4 mentions)
Cfx96 real time system, by Bio-Rad (4 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
148 protocols using qiashredder spin column
1
Quantitative Analysis of B Cell Subsets
2
Cell Monolayer Lysis and RNA Extraction
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In brief, cell monolayers were washed with PBS and lysed using the RLT lysis buffer from the RNeasy minikit (Qiagen, UK). A cell scraper was used to detach cells from the culture flask and the resulting lysate transferred to a QIAshredder spin column (Qiagen) for homogenisation. The QIAshredder column was centrifuged for 2 minutes at 13200 RPM, the column removed and the lysate stored at -80°C until required.
3
Embryo RNA Extraction and Quantification
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Embryos were dechorionated in 50% bleach and washed three times with phosphate-buffered saline (PBS) with 0.1% Tween 20 (PBS-T). Embryos were preserved in RNAlater (Invitrogen) and macerated in 50 μl of RLT buffer (QIAGEN RNeasy Plus Micro kit) using a precooled 1-ml Dounce homogenizer. The lysate was passed through a QIAshredder spin column (QIAGEN). RNA was then isolated using the QIAGEN RNeasy Plus Micro kit. RNA concentration and quality were determined using a NanoDrop 2000 spectrometer (Thermo Fisher Scientific), Qubit 2.0 fluorometer (Invitrogen), and/or a 2200 TapeStation with High Sensitivity RNA screen tapes (Agilent).
4
RNA Isolation and Quantification for miRNA Studies
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Total RNA was isolated using an RNeasy kit (Qiagen) following the manufacturer's protocol. RNA yields were measured using RiboGreen fluorescent dye (Molecular Probes, Eugene, OR, USA). Briefly, HTM cells were transfected with miR-183 mimic (183M) or scramble control mimic (ConM), using the Nucleofector system (Amaxa, Inc.). Three days after transfection, HTM cells were harvested by adding lysis buffer from the kit and homogenization using a QIAshredder spin column (Qiagen). DNA was removed using DNase I (Qiagen). RNA was selectively isolated from the column flow through use of an RNeasy mini-spin column (Qiagen). Twenty microliters of RNase-free water was added to the spin column to recover RNA for each sample. The RNA quantity was analyzed using the RNA Nanodrop with the Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA).
5
RNA Extraction and Allele Quantification
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Embryos were lysed in RLT buffer (Qiagen) supplemented with 0.01% 2-mercaptoethanol. After two rounds of vortexing (15 s each), lysates were applied directly to a QIAshredder spin column (Qiagen) and centrifuged for 3 min at >15,000 g . RNA was extracted using the RNAeasy Mini kit, including DNase treatment, and following the manufacturer's instructions. RNA samples were systematically run on an agarose gel to check their integrity. cDNA was prepared as described above for the gene expression analysis of mESCs, and was then PCR amplified with biotinylated primers and pyrosequenced for allele quantification on a Pyromark Q24 system (Qiagen). The same PCR approach was performed on no-reverse transcription control samples to confirm the absence of genomic DNA contamination. The primers used were designed with PyroMark Assay Design software and validated on XX polymorphic genomic DNA at a ratio of 50:50% (±4%). A list of primers and SNPs used for allele quantification can be found in Galupa et al. (2020) (link).
6
RNA Extraction from Frozen Samples
7
Microarray Analysis of Extracted RNA
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Total RNA was extracted from three replicates using a QIA shredder spin column (Catalog no. 79654, Qiagen) as published previously34 (link). The RNeasy mini kit (Qiagen) was used to purify the extracted RNA. Microarray analysis was done using CodeLink Whole Genome Bioarrays representing 55,000 probes. Scanned images from arrays (gridding and feature intensity) were processed with the CodeLink Expression Analysis software (GE Healthcare), and the data generated for each feature on the array were analyzed with GeneSpring software (Agilent Technologies). Raw intensity data for each gene on every array were normalized to the median intensity of the raw values from that array.
8
Mushroom DNA Extraction Protocol
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After collection, DNA was isolated from the fresh caps using the DNeasy plant tissue kit (Qiagen). Isolations were carried out as described by the supplier. In brief, 100 mg of each mushroom cap was measured and homogenized with a pestle. A total of 400 μl AP1 buffer and 4 μl RNase A were added to a 1.5 ml Eppendorf tube. The samples were vortexed and incubated under rotation for 10 min at 65°C. After the incubation, 130 μl of P3 buffer was added and samples were incubated on ice for 5 min. Subsequently, samples were centrifuged for 5 min at 20,000g and supernatants were transferred to a Qiashredder spin column (Qiagen). Samples were then recentrifuged at 20,000g for 2 min. The flow‐through was transferred to a clean tube. A total of 1.5 volumes of buffer AW1 was added and 650 μl of this mixture was transferred to a DNeasy Mini spin column (Qiagen). Further isolation was exactly as described by the manufacturer.
9
Western Blot Analysis of Respiratory Syncytial Virus
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Infected Vero cells from single-cycle infections were harvested in 1X NuPage LDS sample buffer (Thermofisher) diluted in PBS containing protease inhibitor (Roche). Cell lysates were homogenized using a QIAshredder spin column (Qiagen) and protein concentrations were determined by BCA assay (Pierce BCA Protein Assay kit). Thirty μg of proteins was denatured in a final composition of 1X NuPage LDS sample buffer (ThermoFisher) and 1X NuPAGE Sample Reducing Agent (Invitrogen) by heating at 90°C for 10 min before being subjected to electrophoresis on NuPAGE 4–15% Mini-PROTEAN TGX Gels (Bio-Rad) with 1X TGS Running Buffer (Bio-Rad). Odyssey Protein Molecular Weight Marker (Li-Cor) was run in parallel. Proteins were transferred to PVDF membranes using the iBlot 2 Gel Transfer Device (ThermoFisher) and stained with a primary mouse anti-RSV F (abcam, ab43812), a mouse anti-RSV P (abcam, ab94965) or a mouse anti-RSV G (abcam, 94966) antibody. The rabbit anti-Tubulin (abcam, ab52866n) antibody was used as a loading control. The secondary antibodies used were goat anti-rabbit IgG IRDye 680RD (at 1:15,000, Li-Cor, 926–68071), and goat anti-mouse IgG IRDye 800CW (1:10,000, Li-Cor, 926–32210). Membranes were scanned using Odyssey software, version 3.0 (Li-Cor).
10
Quantifying Transcriptional Changes via qPCR
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To examine transcriptional changes in gene expression, RNA was extracted with the miRNeasy Micro kit (217004; Qiagen; Valencia, CA) according to manufacturer’s protocol with slight modifications as follows. Cells were suspended in QIAzol lysis reagent and loaded onto a QIAshredder spin column (79654; Qiagen) for consistent homogenization after which the RNA was extracted as recommended and the concentration/purity was determined on a Nanodrop2000c (Thermo Fisher Scientific).
Transcriptional changes were determined with real-time quantitative polymerase chain reaction (qPCR) using TaqMan assays (S1 Table ). RNA was reverse transcribed using the High Capacity cDNA reverse transcription kit (4368813, Thermo Fisher Scientific). qPCR was performed on a QuantStudio 6 Flex (Thermo Fisher Scientific) using the conditions recommended by the manufacturer.
Transcriptional changes were determined with real-time quantitative polymerase chain reaction (qPCR) using TaqMan assays (
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