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Rlt rna lysis buffer

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The RLT RNA lysis buffer is a reagent used in the RNA extraction and purification process. It functions to disrupt cells and denature RNases, thereby releasing and protecting RNA from degradation. The buffer is a core component in RNA isolation workflows, facilitating the efficient extraction of high-quality RNA samples for various downstream applications.

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Lab products found in correlation

Rneasy mini kit, by Qiagen (3 mentions) Ripa western lysis buffer, by Thermo Fisher Scientific (2 mentions) Bovine collagen 1, by Advanced BioMatrix (2 mentions) Tnf α elisa kit, by R&D Systems (1 mentions) Dulbecco modified eagle medium (dmem), by GE Healthcare (1 mentions) Amphotericin b, by GE Healthcare (1 mentions) L glutamine, by GE Healthcare (1 mentions) Gentamicin, by GE Healthcare (1 mentions) Nuclear extraction kit, by Active Motif (1 mentions) Pen strep, by GE Healthcare (1 mentions) Transam nf κb p65 assay kit, by Active Motif (1 mentions) Recombinant human il 13, by R&D Systems (1 mentions) Recombinant human il 13 protein, by R&D Systems (1 mentions) Smart seq v4 ultra low input rna kit for sequencing, by Takara Bio (1 mentions) Ion s5, by Thermo Fisher Scientific (1 mentions) β2 mercaptoethanol, by Merck Group (1 mentions) Histopaque 1077, by Merck Group (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Skbr3, by American Type Culture Collection (1 mentions) Hs578t, by American Type Culture Collection (1 mentions)

Spelling variants of this product

RLT buffer (1835 citations) RLT lysis buffer (402 citations) Lysis buffer (117 citations) RNA lysis buffer (37 citations) RLT-plus RNA lysis buffer (6 citations) Buffer RLT lysis buffer (5 citations) RNA RLT buffer (2 citations) Buffer RLT RNA lysis solution (2 citations)

14 protocols using rlt rna lysis buffer

1

Bronchial Epithelial Cell Culture and Analysis

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Bronchial epithelial cell growth medium (BEGM) with antibiotics was purchased from Lonza (Walkersville, MD). The BEGM was prepared following manufacturer’s guideline, which contained all the supplements (BPE, hEGF, epinephrine, transferrin, insulin, retinoic acid, triiodothyronine, GA) except hydrocortisone to avoid any inhibitory effect of corticosteroids on cell pro-inflammatory responses. RNA lysis buffer RLT was from Qiagen (Hilden, Germany). RIPA western lysis buffer was purchased from Thermo-Fisher Scientific (Waltham, MA). DMEM (high glucose) for making D10 (DMEM + 10% FBS + 1% Pen/Strep + 1% Amphotericin B + 1% L-Glutamine+ 0.5% Gentamicin) was from GE Life Sciences (Logan, UT). The nuclear extraction kit and TransAM NF-κB p65 assay kit were from Active Motif (Carlsbad, CA). IL-8, IP-10 and TNF-α ELISA kits were obtained from R&D systems (Minnieaplois, MN).
Mubarak R.A., Roberts N., Mason R.J., Alper S, & Chu H.W. (2018). Comparison of pro- and anti-inflammatory responses in paired human primary airway epithelial cells and alveolar macrophages. Respiratory Research, 19, 126.
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2

Fibroblast Cytokine Response to Rhinovirus

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NIH 3T3 fibroblasts were from The American Type Culture Collection (ATCC). Transwells were purchased from Corning Incorporated, Coring, NY. Bovine collagen I was obtained from Advanced BioMatrix (Carlsbad, CA). Submerged cell culture media consisted of Bronchial Epithelial Cell Growth Medium (BEGM) with antibiotics from Lonza (Walkersville, MD). Air–liquid interface (ALI) media consisted of the PneumaCult kit purchased from Stem Cell Technologies (Vancouver Canada). Recombinant human IL-13 from R&D systems, Minneapolis, MN was reconstituted in 0.1% bovine serum albumin (BSA) aliquots and stored at − 80 °C. Human rhinovirus 16 (HRV16) was propagated in H1-Hela cells (ATCC) and purified as previously described [8 (link)]. RNA lysis buffer (RLT) was from Qiagen (Hilden, Germany). IL-8 and IP-10 ELISA kits were purchased from R&D systems (Minneapolis, MN).
Roberts N., Al Mubarak R., Francisco D., Kraft M, & Chu H.W. (2018). Comparison of paired human nasal and bronchial airway epithelial cell responses to rhinovirus infection and IL-13 treatment. Clinical and Translational Medicine, 7, 13.
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3

Neutrophil Activation Assay and Gene Expression

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MPO activity was measured in flash-frozen liver tissue as previously described (Cassini-Vieira et al., 2015 ). Neutrophils were isolated from bone marrow (Supplemental methods), resuspended in RPMI +10% FBS, and plated in 96-well plates at a density of 2x106 cells per mL. After 1h, allowing cells to acclimate to plating, neutrophils were challenged with or without 100 ng/mL bacterial lipopolysaccharide (#L-2654, Millipore Sigma) for 90 minutes. The plate was briefly spun to pellet cells, the cell-free supernatant was collected to perform the MPO activity assay (#600620, Cayman Chemical, Ann Arbor, MI) per manufacturer’s instructions. RPMI + 10% FBS served as a background control. Both MPO assays were terminated at 5 minutes after substrate addition. Activity was determined by change in O.D. at 450 nM over 5 minutes.
After removal of supernatant, neutrophils were washed with cold PBS, pelleted by centrifugation and resuspended in RNA lysis buffer RLT (Qiagen, Germantown, MD) for RNA isolation with RNeasy mini kits. Expression of neutrophil-associated mRNA was determined with gene-specific primers listed in Table S1.
Poulsen K., McMullen M., Huang E., Kibler C., Sheehan M., Leng L., Bucala R, & Nagy L. (2019). Novel Role of Macrophage Migration Inhibitory Factor in Upstream Control of the Unfolded Protein Response after Ethanol Feeding in Mice. Alcoholism, clinical and experimental research, 43(7), 1439-1451.
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4

Differentiation of Bronchial Epithelial Cells

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Recombinant human IL-13 protein from the R&D Systems, Minneapolis, MN was reconstituted in 0.1% bovine serum albumin (BSA) and stored at -80°C. Bronchial epithelial cell growth medium (BEGM) with antibiotics was purchased from Lonza, Walkersville, MD, USA. Air-liquid interface culture media (F6 media) consisted of 1:1 ratio of bronchial epithelial basal medium (BEBM) and Dulbecco's modified eagle medium (DMEM) plus insulin, transferrin, epinephrine, bovine pituitary extract (BPE), gentamicin and amphotericin, bovine albumin (0.5 μg/ml, ethanolamine (80 μM), MgCl2 (0.3 mM), MgSO4 (0.4 mM), CaCl2 (1 mM), retinoic acid (30 ng/ml), hEGF (10 ng/ml). Bovine collagen I (3 mg/ml) was obtained from Advanced BioMatrix (San Diego, CA). RNA lysis buffer (RLT) was from Qiagen (Hilden, Germany). RIPA Western lysis buffer was purchased from Thermo-Fisher Scientific (Waltham, MA).
Ito Y., Al Mubarak R., Roberts N., Correll K., Janssen W., Finigan J., Mishra R, & Chu H.W. (2018). IL-13 induces periostin and eotaxin expression in human primary alveolar epithelial cells: Comparison with paired airway epithelial cells. PLoS ONE, 13(4), e0196256.
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5

Infection of Airway Epithelial Cells

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Culture and infection of pBEC and alveolar epithelial cultures at air liquid interface (ALI) was performed as previously described.24 ,25 (link) Briefly, prior to infection, cells were washed once with PBS and inoculated with equal number of virus particles for Delta, Omicron BA.2 and BA.5. Two hours post-incubation at 37 °C virus inoculum was removed and unbound virus was washed off using 500 μL of PBS. At 3 days post-infection cells, were collected in RNA lysis buffer (RLT) (Qiagen) with 1% 2-mercaptoethanol for total RNA extraction.
Akerman A., Milogiannakis V., Jean T., Esneau C., Silva M.R., Ison T., Fichter C., Lopez J.A., Chandra D., Naing Z., Caguicla J., Li D., Walker G., Amatayakul-Chantler S., Roth N., Manni S., Hauser T., Barnes T., Condylios A., Yeang M., Wong M., Foster C.S., Sato K., Lee S., Song Y., Mao L., Sigmund A., Phu A., Vande More A.M., Hunt S., Douglas M., Caterson I., Britton W., Sandgren K., Bull R., Lloyd A., Triccas J., Tangye S., Bartlett N.W., Darley D., Matthews G., Stark D.J., Petoumenos K., Rawlinson W.D., Murrell B., Brilot F., Cunningham A.L., Kelleher A.D., Aggarwal A, & Turville S.G. (2023). Emergence and antibody evasion of BQ, BA.2.75 and SARS-CoV-2 recombinant sub-lineages in the face of maturing antibody breadth at the population level. eBioMedicine, 90, 104545.
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6

Transcriptome Profiling of Mouse Cells

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RNA-sequencing was performed as previously described (10 (link)). Briefly, cells were lysed in RLT RNA lysis buffer (Qiagen) containing 1% 2-mercaptoethanol, followed by RNA reverse transcription by SMART-seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech). After fragmentation of cDNA using the Covaris sonication system, sequencing libraries were prepared using the Kapa Library preparation kit for IonTorrent (KAPA) by following the manufacturer’s protocol. Sequencing of cDNA libraries was performed on a IonS5 (Thermo Scientific). Acquired sequencing results were mapped to the reference mouse genome (mm9) using Tophat2, and unmapped sequences were analyzed again by bowtie2. Normalized FPKM values were acquired using Cuffnorm of the Cufflinks package (version 2.2.1, Trapnell Lab) under default settings. Gene set enrichment analysis (30 (link)) was performed with the following settings: collapse = true, permutation type = gene_set, scoring = weighted, metric = log2_ratio_of_classes. Hierarchical clustering was performed using the heatmap.2 function in R package gplots.
Mikami N., Kawakami R., Chen K.Y., Sugimoto A., Ohkura N, & Sakaguchi S. (2020). Epigenetic conversion of conventional T cells into regulatory T cells by CD28 signal deprivation. Proceedings of the National Academy of Sciences of the United States of America, 117(22), 12258-12268.
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7

Extraction and Purification of Total RNA from Human Neurons

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Total RNA was extracted from human neurons using the RNeasy Mini kit (Qiagen, catalogue number: 74104). Cultures were washed with D‐PBS (ThermoFisher Scientific, catalogue number: 1404017) and lysed with 350 μl of RLT RNA lysis buffer (Qiagen, catalogue number: 79216) containing 1% β‐2‐mercaptoethanol (Sigma‐Aldrich, catalogue number: M6250). Lysates were either frozen at −80 °C or processed with the RNeasy Mini kit according to the manufacturer's instructions, including a DNase treatment for 15 min at room temperature (22°C) (Qiagen, catalogue number: 79254).
Ateaque S., Merkouris S., Wyatt S., Allen N.D., Xie J., DiStefano P.S., Lindsay R.M, & Barde Y. (2022). Selective activation and down‐regulation of Trk receptors by neurotrophins in human neurons co‐expressing TrkB and TrkC. Journal of Neurochemistry, 161(6), 463-477.
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8

Osteogenic Differentiation of DPSCs

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A3 DPSCs (2.2 × 104/cm2) were seeded in six-well plates and treated with DDM liposomes at concentrations of 0–100 μg/mL (lipid concentration), free DDM (10 μg/mL), or osteogenic media (supplemented α-MEM plus 10 nM dexamethasone and 100 μM β-glycerophosphate) for 3, 9, 21, and 35 days. DDM liposomes (DDM concentrations of 0, 1, 10, and 100 μg/mL) were ultracentrifuged, resuspended in supplemented α-MEM and sterile filtered. After treatments, cells were scraped in RLT RNA lysis buffer (Qiagen) for molecular analysis.
Melling G.E., Colombo J.S., Avery S.J., Ayre W.N., Evans S.L., Waddington R.J, & Sloan A.J. (2018). Liposomal Delivery of Demineralized Dentin Matrix for Dental Tissue Regeneration. Tissue Engineering. Part A, 24(13-14), 1057-1065.
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9

Isolation and Culture of PBMCs

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PBMCs were isolated from heparinized blood by using Ficoll-Hypaque gradient centrifugation.5 (link) Cells were lysed immediately in RLT RNA lysis buffer (Qiagen, Valencia, Calif) for analysis of gene expression in freshly isolated cells or cultured in medium (RPMI containing 10% AB+ serum, 50 μg/mL streptomycin, and 50 U/mL penicillin) with 10−8 mol/L FLU or medium without FLU for 3 hours. Cells were then harvested and stored in RNA lysis buffer at −80°C.
Goleva E., Babineau D.C., Gill M.A., Jackson L.P., Shao B., Hu Z., Liu A.H., Visness C.M., Sorkness C.A., Leung D.Y., Togias A, & Busse W.W. (2018). Expression of corticosteroid-regulated genes by PBMCs in children with asthma. The Journal of allergy and clinical immunology, 143(3), 940-947.e6.
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10

PBMC Isolation and RNA Extraction

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The CPT™ tubes were centrifuged at 1600× g for 20 min. The upper plasma layer was discarded, the opaque layers containing PBMCs were combined in a new tube and filled up to a total of 50 mL with PBS, mixed and centrifuged at 300× g for 15 min. The supernatant was discarded, the PBMC pellet washed in PBS and centrifuged again at 300× g for 10 min. The cell pellet was lysed in RLT RNA lysis buffer (Qiagen, Hilden, Germany) containing 1% β-Mercaptoethanol. Blood samples taken in CPT™ tubes from the HD group were processed alike. Cell lysates were kept at −80° C until RNA extraction.
Welsch E., Schuster E., Krainer M., Marhold M., Bartsch R., Fischer M.B., Hermann M., Hastermann G., Uher H., Sliutz G., Anker B., Zeillinger R, & Obermayr E. (2023). Comparison of RNA Marker Panels for Circulating Tumor Cells and Evaluation of Their Prognostic Relevance in Breast Cancer. Cancers, 15(4), 1271.
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