Rna lysis buffer

Manufactured by Thermo Fisher Scientific

The RNA lysis buffer is a reagent used in molecular biology applications to facilitate the lysis, or rupturing, of cells and the subsequent release of RNA. This buffer contains components that disrupt cell membranes and denature cellular proteins, enabling the extraction and purification of RNA from samples.

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20 protocols using rna lysis buffer

Isolation and Treatment of Cancer-Associated Fibroblasts

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Transplanted tumors were harvested at the indicated time points to prepare single-cell suspensions as previously described. After red blood cell lysis and blocking of Fc receptors, cells were stained with anti-PDGFRα and anti-F4/80 antibodies (Biolegend) at a 1:100 dilution for 30 min on ice. CAFs (PDGFRα⁺F4/80^-) were sorted by Fluorescence Activated Cell Sorting (FACS) on an Aria II cell sorter (BD Biosciences) 15 . Sorted CAFs were suspended in RNA lysis buffer (Invitrogen) or seeded in 24-well plates in DMEM containing 10% FBS and 100 units/ml penicillin/streptomycin. Subsequently, non-adherent cells were removed by extensive washing with PBS and adherent cells were treated with recombinant mouse Fgl2 (1 µg/ml or 5 µg/ml, R&D Systems) for further analysis.

Zhu Y., Zhang L., Zha H., Yang F., Hu C., Chen L., Guo B, & Zhu B. (2017). Stroma-derived Fibrinogen-like Protein 2 Activates Cancer-associated Fibroblasts to Promote Tumor Growth in Lung Cancer. International Journal of Biological Sciences, 13(6), 804-814.

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Isolation of cells from hydrogels

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To retrieve cells, hydrogels were incubated at 37 °C with 250 μL of digestion solution containing 34 U mL⁻¹ alginate lyase (Sigma-Aldrich) and 300 U mL⁻¹ collagenase type I (Sigma-Aldrich). After 20 min, the hydrogels were mechanically disrupted by pipetting and an extra 250 μL of fresh digestion mixture was added. The gels were incubated at 37 °C for an additional 20 min.
Next, 0.1 mL of wash buffer (DPBS w/o Ca/Mg, 2 mM EDTA, 0.5 % BSA) was added and the entire mixture was transferred from the culture plates to low protein binding Eppendorf tubes and centrifuged at 400 g for 5 min at 4 °C. The supernatant was discarded and two additional washes were performed. The cell number and viability were assessed using a benchtop cell analyzer (MUSE), before the addition of RNA lysis buffer (Invitrogen), supplemented with 1 % β-mercaptoethanol. After vortexing, samples were stored at −80 °C until use.

Gonzalez-Pujana A., Vining K.H., Zhang D.K., Santos-Vizcaino E., Igartua M., Hernandez R.M, & Mooney D.J. (2020). Multifunctional biomimetic hydrogel systems to boost the immunomodulatory potential of mesenchymal stromal cells. Biomaterials, 257, 120266.

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Mouse Retina and RPE Isolation

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For the collection of mouse retina and RPE, the isolated eyes were placed in the cold petri dish under a surgical microscope. With careful precision, the anterior parts, including the cornea, lens, and iris, as well as connective tissues, muscles, and optic nerve, were excised. Radial cuts were made symmetrically in a four‐leaf pattern, allowing for the careful removal of the neural retina from the mouse eyecup. The pigmented RPE/choroid layer was gently scraped from the sclera. RPE/choroid and neural retina tissues were immediately transferred to ice‐cold 1X RIPA buffer (Thermo Scientific 89,900, Rockford, IL) for cell lysate preparation, 1X pre‐lysis buffer (Epigentek OP‐0006, Farmingdale, NY) for histone extract preparation, or RNA lysis buffer (Invitrogen 12183016, Carlsbad, CA) for RNA isolation, based on the specific experimental requirements. The tissues were snap‐frozen and stored in liquid nitrogen to keep them intact if not used immediately.

Dubey S.K., Dubey R., Prajapati S.C., Jung K., Mohan K., Liu X., Roney J., Tian W., Abney J., Giarmarco M.M., Hernandez A.G., Liu J, & Kleinman M.E. (2024). Histone deficiency and hypoacetylation in the aging retinal pigment epithelium. Aging Cell, 23(5), e14108.

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Fibroblast Response to Plasma Treatment

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Fibroblasts cells in a 6-well plate were grown for 48 hours at 37°C. The growth medium was removed and fresh growth medium with either 2% human control plasma or 2% human trauma plasma was added to each well. The cells were then incubated for 6 hours, 24 hours and 48 hours at 37°C. At the end of incubation time, the cells were gently scraped from the wells and added to RNA lysis buffer (Invitrogen, Carlsbad, CA) for RNA isolation.

Miller E.S., Loftus T.J., Kannan K.B., Plazas J.M., Efron P.A, & Mohr A.M. (2019). Systemic regulation of bone marrow stromal cytokines following severe trauma. The Journal of surgical research, 243, 220-228.

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Aortic Valve Transcriptional Profiling

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All three aortic valve leaflets were carefully excised from the heart and placed in RNA lysis buffer (Invitrogen)/1% β-mercaptoethanol (Sigma). Isolation of RNA was performed using Invitrogen spin-columns. After conversion of RNA to cDNA (VILO reverse transcriptase, Invitrogen), quantitative real-time PCR was performed on a StepOnePlus RT-PCR machine. TaqMan Gene Expression Assay primers were used to assess mRNA levels for genes of interest. A full table of primers used is provided in the Supplementary Table 2. All genes were normalized by hypoxanthine phosphoribosyltransferase 1 (HPRT1), a commonly used housekeeper gene, and values are expressed via the ΔΔCT method.

Roos C.M., Zhang B., Hagler M.A., Verzosa G.C., Huang R., Oehler E.A., Arghami A, & Miller J.D. (2021). Effects of Altering Mitochondrial Antioxidant Capacity on Molecular and Phenotypic Drivers of Fibrocalcific Aortic Valve Stenosis. Frontiers in Cardiovascular Medicine, 8, 694881.

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Flow Cytometric Analysis of Adipose B Cells

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Isolated cells were incubated with Ghost Dye Red 780 Viability Dye (TONBO biosciences) for 30 minutes on ice, in the dark, then washed and stained with FcBlock and surface antibodies for 45 minutes to 12 hours at 4°C in the dark. Antibodies used are documented in Table S1. For intracellular staining of cytokines, cells were treated with GolgiStop (containing monensin) for 4 hours and fixed for 20 minutes using fixation/permeabilization solution (BD, 554715) prior to intracellular staining for 45 minutes. Analysis was performed on a BD Fortessa X-30 and BD FACSSymphony A3 cytometers and using FlowJo v10; gating schemes are shown in relevant supplementary figures. Antibodies used can be found in Table S1. For fluorescence associated cell sorting: 15,000 live CD45⁺ CD19⁺ CD11b⁺ and live CD45⁺ CD19⁺ CD11b⁻ were sorted on a BD FACS Aria II into RPMI with 40% FBS. Cells were pelleted and resuspended in RNA lysis buffer (Invitrogen; 46-6001) for RNA isolation. Adipose B cells were pooled from 2 mice per sample to account for biological variability.

Carey A., Nguyen K., Kandikonda P., Kruglov V., Bradley C., Dahlquist K.J., Cholensky S., Swanson W., Badovinac V.P., Griffith T.S, & Camell C.D. (2024). Age-associated accumulation of B cells promotes macrophage inflammation and inhibits lipolysis in adipose tissue during sepsis. Cell reports, 43(3), 113967.

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RNA Isolation and RT-qPCR Analysis

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Cells were lysed in RNA Lysis buffer (Ambion) supplemented with β-Mercaptoethanol and passed through Qiashredder columns (Qiagen). RNA was then isolated using Pure l Link RNA Mini Kit columns (Ambion) following the manufacturer’s recommendations. RNA samples were treated with DNAseI (ThermoScientific) for 30 min at 37°C followed by 10 min EDTA deactivation at 65°C. RT-qPCR was carried out with a Bio-Rad CFX384 real time cycler with Taqman probes as directed by the manufacturer. Data were normalized against the expression of a control gene (GAPDH) followed by the uninduced or uninfected (Control-Ctrl) sample (set to 1). When no counts were detected in a sample, an arbitrary number of 45 cycles was selected.

Odendall C., Voak A.A, & Kagan J.C. (2017). Type III interferons are commonly induced by bacteria-sensing TLRs, and reinforce epithelial barriers during infection. Journal of immunology (Baltimore, Md. : 1950), 199(9), 3270-3279.

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RNA Sequencing of Adult and Fetal Tissues

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Human adult and fetal tissue samples were mechanically homogenized using a hand-held homogenizer (Thermo Fisher Scientific) and lysed in 500 μl RNA lysis buffer (Ambion) by sonication (1 × 2–4 s pulse, Branson SFX150). RNA was isolated from 3 × 30 μm sections of human adult and fetal tissue using the RNAqueous Micro Kit (Ambion), and total RNA samples were DNase-treated (Ambion). Sample yield and integrity was analyzed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA sequencing was performed by standard operating procedure by GENEWIZ (https://www.genewiz.com/en) using Illumina HiSeq with a 2 × 150 bp configuration. Quality control of the obtained sequences was performed using FastQC (Wingett and Andrews, 2018 (link)) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ Accessed 12/10/2017). The results were further reviewed by MultiQC (Ewels et al., 2016 (link)). Adaptor sequences, low-quality bases from both sides of the read (3 bases or smaller), and reads with a length smaller than 36 bp were discarded by Trimmomatic (Bolger et al., 2014 (link)).

Saitou M., Gaylord E.A., Xu E., May A.J., Neznanova L., Nathan S., Grawe A., Chang J., Ryan W., Ruhl S., Knox S.M, & Gokcumen O. (2020). Functional Specialization of Human Salivary Glands and Origins of Proteins Intrinsic to Human Saliva. Cell reports, 33(7), 108402.

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RPE Cell Activation by IL-33 and LPS

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RPE cell line (B6-RPE07) was derived from a 12-week-old naïve female C57BL/6 mouse and generated as described previously 28 (link). Cells were cultured in T75 flasks in complete DMEM containing 10% (v/v) fetal calf serum (Sigma) and incubated in 5% CO₂ at 37°C. Seventy percent confluent cells were detached from flasks using 0.25% trypsin-EDTA and cultured in 24-well plates with different concentrations of IL-33 (10–100 ng/mL) or LPS (100 ng/mL, Sigma) for 24 h. Cells were then collected and lysed with RNA lysis buffer (Ambion, Life Technologies) for analysis of Il33 and Mcp-1 mRNA expression using Fast SYBR® Green real time PCR.

Barbour M., Allan D., Xu H., Pei C., Chen M., Niedbala W., Fukada S.Y., Besnard A.G., Alves-Filho J.C., Tong X., Forrester J.V., Liew F.Y, & Jiang H.R. (2014). IL-33 attenuates the development of experimental autoimmune uveitis. European Journal of Immunology, 44(11), 3320-3329.

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Profiling PBMC Transcriptome Responses

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Thawed PBMC were rested overnight and left unstimulated or stimulated with iRBC for 4 hours. Cells were promptly stained for cell surface markers, and ~1 × 10⁴ to 3 × 10⁴ cells (>99% pure) were double-sorted directly into RNA lysis buffer (Ambion) with a FACSAria (BD) and stored at −80 until RNA amplification.

Jagannathan P., Kim C.C., Greenhouse B., Nankya F., Bowen K., Eccles-James I., Muhindo M.K., Arinaitwe E., Tappero J.W., Kamya M.R., Dorsey G, & Feeney M.E. (2014). Loss and dysfunction of Vδ2+ γδ T cells is associated with clinical tolerance to malaria. Science translational medicine, 6(251), 251ra117.

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