Rlt lysis buffer

Manufactured by Qiagen

Sourced in Germany, United States, Netherlands, United Kingdom, France, Australia

RLT lysis buffer is a guanidine-thiocyanate-containing lysis buffer designed for efficient lysis and homogenization of biological samples to facilitate subsequent RNA extraction and purification.

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Lab products found in correlation

Rneasy mini kit, by Qiagen (87 mentions) Rneasy micro kit, by Qiagen (35 mentions) β mercaptoethanol, by Merck Group (20 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (18 mentions) Rneasy kit, by Qiagen (17 mentions) Rneasy plus mini kit, by Qiagen (12 mentions) Rnase free dnase set, by Qiagen (10 mentions) Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (10 mentions) 2100 bioanalyzer, by Agilent Technologies (9 mentions) Tissuelyser, by Qiagen (8 mentions) Rnalater, by Qiagen (8 mentions) Qiashredder, by Qiagen (8 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (8 mentions) Nanodrop, by Thermo Fisher Scientific (8 mentions) Allprep dna rna mini kit, by Qiagen (7 mentions) Rneasy rna isolation kit, by Qiagen (7 mentions) Lysing matrix e tube, by MP Biomedicals (7 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (7 mentions) Rnalater, by Thermo Fisher Scientific (7 mentions) Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (7 mentions)

Close spelling variants of this product

RLT buffer (1835 citations) Lysis buffer (117 citations) RLT Plus lysis buffer (45 citations) RLT RNA lysis buffer (14 citations) RNeasy lysis buffer RLT (11 citations) RLT cell lysis buffer (8 citations) RLT-plus RNA lysis buffer (6 citations) Buffer RLT lysis buffer (5 citations) Buffer RLT RNA lysis solution (2 citations) RLT-beta-mercaptoethanol 1/100 lysis buffer (2 citations)

402 protocols using rlt lysis buffer

Quantifying Csf2-expressing Cell Populations

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For quantitative PCR (qPCR) analysis of major Csf2-expressing cell populations (Fig. 1 H), Csf2-tdTomato⁺ cells were sorted from homogenized lungs that were pooled from two or three individual Csf2^+/fl P10 mice. The three cell populations collected were ILC2s (tdTomato⁺CD45⁺Lin⁻CD3⁻Thy1.2⁺ST2⁺), T cells (tdTomato⁺CD45⁺CD3⁺), and epithelial cells (tdTomato⁺CD45⁻EpCAM⁺). Cells were sorted directly into RLT Lysis Buffer (Qiagen) using a FACSAria III (BD Biosciences). Each channel was loaded with 5,000–100,000 cells from each sample.
To verify recombination of the Csf2^fl allele in ILC2s by qPCR (Fig. S2 A), ILC2s (CD45⁺Lin⁻CD3⁻Thy1.2⁺ST2⁺) were sorted from homogenized P10 lungs of Csf2^fl and Vav1^iCre/+;Csf2^fl mice. Cells were sorted directly into RLT Lysis Buffer (Qiagen) using a FACSAria III (BD Biosciences). Each channel was loaded with 15,000–30,000 cells per sample.
For PCR analysis of the Csf2^Δ allele (Fig. S2 B), CD45⁺ cells were sorted from homogenized P10 lungs of Csf2^fl and Vav1^iCre/+;Csf2^fl mice. 600,000 cells were sorted into RPMI-1640 using a FACSAria III (BD Biosciences). The cells were then digested, and the recombination state of the Csf2^fl allele was detected by standard PCR (Kapa Hotstart Mouse Genotyping Mix; Sigma; KK7352) using the same primer combination as for Csf2^Δ genotyping listed above.

Gschwend J., Sherman S.P., Ridder F., Feng X., Liang H.E., Locksley R.M., Becher B, & Schneider C. (2021). Alveolar macrophages rely on GM-CSF from alveolar epithelial type 2 cells before and after birth. The Journal of Experimental Medicine, 218(10), e20210745.

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BMSC Transcriptional Profiling Workflow

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Post-sorting, 200,000 BMSCs per sample were spun down and treated on ice with RLT lysis buffer (Qiagen, Hilden, Germany) with 1% β-mercaptoethanol. BMSCs in monolayer were washed with PBS and treated on ice with RLT lysis buffer (Qiagen) with 1% β-mercaptoethanol. A range of 0.25–1.00 µg of purified RNA (RNeasy Micro Kit; Qiagen) was reverse transcribed into cDNA (RevertAid First Strand cDNA Synthesis Kit; MBI Fermentas, St. Leon-Rot, Germany). RT-PCR was performed using an annealing temperature of 60 °C on a C1000 Touch™ Thermal Cycler using SybrGreen (Eurogentec, Seraing, Belgium). The data were normalized to the housekeeper gene RSP27a. The relative expression was calculated according to the 2^−ΔΔCt formula. The primers used for RT-PCR are listed in (Table S1).

Voskamp C., van de Peppel J., Gasparini S., Giannoni P., van Leeuwen J.P., van Osch G.J, & Narcisi R. (2019). Sorting living mesenchymal stem cells using a TWIST1 RNA-based probe depends on incubation time and uptake capacity. Cytotechnology, 72(1), 37-45.

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Isolation and Characterization of HIV-1 RNA from Plasma, PBMC, and Virus Outgrowth Assay

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Plasma was obtained following centrifugation of blood ethylenediaminetetraacetic acid-collection tubes. Peripheral blood mononuclear cells (PBMCs) were separated by gradient centrifugation in cell preparation tubes. CD4⁺ T cells were isolated from PBMCs using a CD4⁺ T-cell negative isolation kit and magnetic-activated cell sorting columns (Miltenyi Biotec, Teterow, Germany; purity >95%). One million CD4⁺ T cells were lysed in QIAgen lysis (RLT) buffer, and the lysates were stored at −80 °C until the RNA and DNA were extracted using the QIAgen AllPrep DNA/RNA Mini Kit (Cat.no. 80204, QIAgen, Melbourne, Australia). During RNA purification, RNA columns were treated twice with QIAgen RNase-Free DNase. Plasma HIV-1 RNA for sequencing was extracted from 2 to 3 ml of plasma by ultracentrifugation and a guanidinium-based method, including a prespin to sediment potential cell remnants [10 (link)]. HIV-1 RNA was extracted from 10 to 100 μl VOA supernatant using the same method as the plasma samples, but without the prespin. From cell-associated HIV-1 RNA, plasma HIV-1 RNA and VOA HIV-1 RNA, we generated complementary DNA using the Superscript III (Invitrogen by Thermo Fisher Scientific) cDNA synthesis kit and a gene-specific primer for p24-RT according to the manufacturer's instructions. See Supplemental Digital Content 2 for primers and PCR conditions.

Winckelmann A., Morcilla V., Shao W., Schleimann M.H., Hojen J.F., Schlub T.E., Benton P.W., Østergaard L., Søgaard O.S., Tolstrup M, & Palmer S. (2018). Genetic characterization of the HIV-1 reservoir after Vacc-4x and romidepsin therapy in HIV-1-infected individuals. AIDS (London, England), 32(13), 1793-1802.

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Quantification of HIV-1 RNA in Plasma, Cells, and Viral Outgrowth

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Plasma was obtained following centrifugation of blood ethylenediaminetetraacetic acid-collection tubes. Peripheral blood mononuclear cells (PBMCs) were separated by gradient centrifugation in cell preparation tubes. CD4 þ T cells were isolated from PBMCs using a CD4 þ T-cell negative isolation kit and magnetic-activated cell sorting columns (Miltenyi Biotec, Teterow, Germany; purity >95%). One million CD4 þ T cells were lysed in QIAgen lysis (RLT) buffer, and the lysates were stored at À80 8C until the RNA and DNA were extracted using the QIAgen AllPrep DNA/RNA Mini Kit (Cat.no. 80204, QIAgen, Melbourne, Australia). During RNA purification, RNA columns were treated twice with QIAgen RNase-Free DNase. Plasma HIV-1 RNA for sequencing was extracted from 2 to 3 ml of plasma by ultracentrifugation and a guanidinium-based method, including a prespin to sediment potential cell remnants [10] . HIV-1 RNA was extracted from 10 to 100 ml VOA supernatant using the same method as the plasma samples, but without the prespin. From cell-associated HIV-1 RNA, plasma HIV-1 RNA and VOA HIV-1 RNA, we generated complementary DNA using the Superscript III (Invitrogen by Thermo Fisher Scientific) cDNA synthesis kit and a gene-specific primer for p24-RT according to the manufacturer's instructions. See Supplemental Digital Content 2 for primers and PCR conditions, http:// links.lww.com/QAD/B287.

Genetic characterization of the HIV-1 reservoir after Vacc-4x and romidepsin therapy in HIV-1-infected individuals: Erratum.. (2018). AIDS (London, England), 32(17).

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Extraction and Quantification of Sinonasal Tissues

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Anterior ethmoid biopsies (4.0 mm²) were obtained intraoperatively per the standard of care and only if the study participant’s (CRSwNP and control subjects) endoscopic sinus surgical procedure involved the excising of tissues from the ethmoid sinuses that would have otherwise been discarded. Sinonasal tissues were immediately snap-frozen and stored at −80°C in 2.0-mL Eppendorf tubes (Fisher Scientific; Waltham, MA) until analyzed. Sinonasal tissues were then thawed on ice, transferred to 0.6 mL of lysis RLT buffer (Qiagen; Hilden, Germany) containing stainless-steel beads (5 mm), and homogenized using a Qiagen Tissuelyser LT. Nucleic acid was extracted using a Qiagen AllPrep RNA microRNA Universal Kit and a QiaCube, followed by subsequent quantification with a NanoDrop 8000 and a Qubit (Thermo Fisher Scientific; Pittsburg, PA), according to manufacturer’s instructions.

Blight B.J., Gill A.S., Sumsion J.S., Pollard C.E., Ashby S., Oakley G.M., Alt J.A, & Pulsipher A. (2021). Cell Adhesion Molecules are Upregulated and May Drive Inflammation in Chronic Rhinosinusitis with Nasal Polyposis. Journal of Asthma and Allergy, 14, 585-593.

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RNA Extraction and Sequencing from Organs

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Organs (OB, BRN, CBL, BST, STM, LIV, and iBAT) were homogenized in Lysis RLT Buffer (Qiagen) supplemented with 1% β-mercaptoethanol (Sigma–Aldrich) by using the OMNI tissue homogenizer (OMNI International). Homogenized organ lysates were then loaded onto the QIAshredder homogenizer spin columns (Qiagen) for further homogenization and elimination of insoluble debris. Total RNA was extracted by using the RNeasy Mini Kit (Qiagen), according to the manufacturer's protocol. White adipose tissues (pgWAT and psWAT) were homogenized in Qiazol (Qiagen) and RNA extracted with the Lipid RNeasy Lipid Tissue Mini Kit (Qiagen). mRNA was prepared for sequencing by using the TruSeq stranded mRNA sample preparation kit (Illumina), with a selected insert size of 120–210 bp. All samples were sequenced on an Illumina HiSeq 4000, generating paired-end 150 bp sequencing reads, and had an average depth of 39.98 ± 1.05 (SEM) million reads (Additional file 2: Table S1).

Huang S.S., Makhlouf M., AbouMoussa E.H., Ruiz Tejada Segura M.L., Mathew L.S., Wang K., Leung M.C., Chaussabel D., Logan D.W., Scialdone A., Garand M, & Saraiva L.R. (2020). Differential regulation of the immune system in a brain-liver-fats organ network during short-term fasting. Molecular Metabolism, 40, 101038.

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RNA-seq of Mouse Olfactory Mucosa

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Animal experiments were carried out under the authority of a UK Home Office license (80/2472), after review by the Wellcome Trust Sanger Institute Animal Welfare and Ethical Review Board. All mice were housed in single sex groups within individually ventilated cages, with access to food and water ad libitum. All WOM samples were obtained from a single animal, except the pup WOM samples, which were the pool of three or four individuals. Details of the strain, age and sex of each animal sequenced can be found in Supplementary file 1. MOEs were dissected and immediately homogenized in lysis RLT buffer (Qiagen, Germantown, Maryland). Total RNA was extracted using the RNeasy mini kit (Qiagen) with on-column DNAse digestion, following the manufacturer’s protocol. mRNA was prepared for sequencing using the TruSeq RNA sample preparation kit (Illumina, San Diego, California). All RNA sequencing was paired-end and produced 100-nucleotide-long reads.

Ibarra-Soria X., Nakahara T.S., Lilue J., Jiang Y., Trimmer C., Souza M.A., Netto P.H., Ikegami K., Murphy N.R., Kusma M., Kirton A., Saraiva L.R., Keane T.M., Matsunami H., Mainland J., Papes F, & Logan D.W. (2017). Variation in olfactory neuron repertoires is genetically controlled and environmentally modulated. eLife, 6, e21476.

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Fractalkine Regulation of BDNF in Microglia

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MG6 cells, a mouse microglia cell line (RCB 2403, RIKEN Cell Bank, Tsukuba, Japan) were maintained in a growth medium composed of Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum supplemented with 100 μM β-mercaptoethanol, 10 μg/ml insulin, 100 μg/ml streptomycin and 100 U/ml penicillin in 90-mm Petri dishes (Thermo Fisher Scientific Inc., Waltham, MA) at 37 °C in 5% CO₂ and 95% air^{55 (link),56 (link)}. The MG6 cells were replated in 6-well plates at a density of 1 × 10⁵ cells/ml in normal DMEM medium. After 4 days, MG6 cells were used in the experiment. To examine the effects of fractalkine on BDNF mRNA expression, fractalkine (100 nM) was applied to the MG6 cells for 3 h. The treated cells were collected with Lysis RLT buffer (QIAGEN, Venlo, Netherlands) containing 1% β-mercaptoethanol for analysis using real-time reverse transcription polymerase chain reaction (RT-PCR).

Kawamura N., Katsuura G., Yamada-Goto N., Nakama R., Kambe Y., Miyata A., Furuyashiki T., Narumiya S., Ogawa Y, & Inui A. (2022). Brain fractalkine-CX3CR1 signalling is anti-obesity system as anorexigenic and anti-inflammatory actions in diet-induced obese mice. Scientific Reports, 12, 12604.

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Efficient Isolation of Bone Marrow RNA

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Flush and centrifugation techniques are commonly used to separate bone and bone marrow in long bones harvested from rodents (Dobson et al., 1999 (link)). Since these approaches are less convenient for transiliac bone biopsies, we developed a specific protocol. The transiliac bone biopsies were centrifuged in nested RNase free centrifugal tubes (Fig. 1). The inner tube (1,5 ml RNase free tube, Biosphere SafeSeal tube, Sarstedt, article number 1050299) was perforated thrice with a sterile 18G needle to allow marrow to elude into the outer tube (2 ml RNase free tube, Sample tubes RB, Qiagen, article number 1050299), containing 600 μl of lysis RLT buffer (Qiagen) with the addition of β-mercaptoethanol, according to the manufacturer's instructions. To reduce the migration time of the bone marrow during centrifugation, biopsies were split in 2 parts. Both parts were placed in the inner tube with the cortex pointing to the top of the tube. Three centrifugation protocols were tested: 1′ at 20,000g; 3′ at 20,000g, and 3′ at 5000g, all at 4 °C. Centrifugation cycles were repeated up to 3 times. All experiments were performed in triplicate (unless otherwise specified).Fig. 1

Road map for isolating bone and bone marrow mRNA from transiliac bone biopsies.

Fig. 1

de Loor H., Smout D., Jørgensen H., Meng C., Van Craenenbroeck A.H, & Evenepoel P. (2022). Isolating mineralized bone and bone marrow mRNA from transiliac bone biopsies stored in a stabilizing solution: A comparative study. Bone Reports, 17, 101624.

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Caco-2 Cell Transcriptome Analysis

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After 2 washes with cold RH buffer, Caco-2 cells were lysed with RLT lysis buffer (Quiagen, Courtaboeuf, France). mRNA extraction was then performed using commercially available RNeasy mini kit (Qiagen, Courtaboeuf, France) in accordance with manufacturer’s instructions. The amount of RNA and the purity were measured via spectrophotometer (absorbance at A260 nm and A280 nm). Reverse transcription (RT) was performed in a final volume of 20 µL containing 250 ng of RNA, RT-MIX (BioRad iScriptTM Reverse Transcription Supermix) and H₂O. The samples were incubated in Thermal Cycler (MJ Research, Hampton, NH, USA) using the following protocol: priming 5 min at 25 °C, reverse transcription 30 min at 42 °C and heating to 85 °C for 5 min to inactivated the reverse transcriptase. Expression levels of transporters studied were evaluated by real-time PCR (qPCR). The templates obtained by the RT were used for qPCR amplifications employing CFX96 Real Time System (Biorad), the Supermix (SsoFastTM EvaGreen Supermix, BioRad) and specific primers listed in Table 2. Specificity and efficacy of each primer was tested before their use. The experimental cycling step condition consisting in an initial enzyme activation at 95 °C for 30”, 39 cycles of denaturation at 95 °C for 5” and annealing/extension step at 60 °C for 5”. β-actin was used as housekeeping gene to normalize mRNA.

Sevin E., Dehouck L., Versele R., Culot M, & Gosselet F. (2019). A Miniaturized Pump Out Method for Characterizing Molecule Interaction with ABC Transporters. International Journal of Molecular Sciences, 20(22), 5529.

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