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Rna lysis buffer

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The RNA lysis buffer is a reagent used in the extraction and purification of RNA from biological samples. Its core function is to lyse cells and disrupt the cellular membranes, releasing the RNA for further processing and analysis.

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Lab products found in correlation

Rneasy mini kit, by Qiagen (5 mentions) Facsaria 2, by BD (3 mentions) Steponeplus machine, by Thermo Fisher Scientific (3 mentions) Rneasy kit, by Qiagen (3 mentions) First strand synthesis kit, by Thermo Fisher Scientific (3 mentions) Rna miniprep kit, by Zymo Research (3 mentions) Sybr reagents, by Thermo Fisher Scientific (3 mentions) Nextseq 500 sequencer, by Illumina (2 mentions) Smart seq v4 ultra low input rna kit, by Takara Bio (2 mentions) Rnaeasy plus micro kit, by Qiagen (2 mentions) Nextera xt dna sample preparation kit, by Illumina (2 mentions) Apc conjugated cd11b mouse clone m1 70, by Miltenyi Biotec (2 mentions) Pe conjugated cd45 antibody, by BD (2 mentions) Cellstar, by Greiner (2 mentions) Rankl tec, by R&D Systems (2 mentions) M csf, by R&D Systems (2 mentions) Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (2 mentions) Sybr green master mix, by Bio-Rad (2 mentions) Rnase free dnase, by Promega (2 mentions) Serial cryostat coronal brain sections, by Leica (2 mentions)

Close spelling variants of this product

Lysis buffer (117 citations) RLT RNA lysis buffer (14 citations) RLT-plus RNA lysis buffer (6 citations) RNeasy RNA Tissue Lysis Buffer Solution (3 citations) Buffer RLT RNA lysis solution (2 citations) AllPrep RNA/DNA FFPE kit lysis buffer (2 citations) RNA lysis buffer (TCL) (2 citations)

37 protocols using rna lysis buffer

1

Multimodal Tumor Tissue Isolation

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Mammary tumors from 4T1, E0771, or EMT6 tumor bearing mice were harvested, and immediately placed in C-Tubes (Miltenyi Biotec) containing fresh DMEM (Life Technologiess) medium with tumor dissociation enzymes from the mouse tumor dissociation kit (Miltenyi Biotec). Tumors were dissociated using the Miltenyi gentleMACS Octo Dissociator with heaters following manufacturers protocol and using the program 37C_m_TDK_2. Following tissue dissociation, cells were washed with 1X PBS with 2% FBS, centrifuged, and red blood cells were lysed in ACK lysis buffer for 30 seconds, followed by two washes in 1X PBS with 2% FBS. All spleens and thymus were harvested and manually dissociated in 1X PBS with 2% FBS using a plunger over a 70 micron strainer (Fisher). Cells were washed and red blood cells were lysed following the protocol outlined above. Additionally, mammary fat pads were excised, flash frozen, manually crushed, and homogenized in RNA lysis buffer (Qiagen) containing 10% BME (Thermo Fisher) for downstream qRT-PCR analysis. Intestines were harvested from mice, and flushed 5 times with 1X PBS to remove waste. Following this, they were flash frozen, manually crushed, and homogenized in RNA lysis buffer (Qiagen) containing 10% BME (Thermo Fisher) for downstream qRT-PCR analysis.
Hinshaw D.C., Benavides G.A., Metge B.J., Swain C.A., Kammerud S.C., Alsheikh H.A., Elhamamsy A., Chen D., Darley-Usmar V., Rathmell J.C., Welner R.S., Samant R.S, & Shevde L.A. (2023). Hedgehog signaling regulates Treg to Th17 conversion through metabolic rewiring in breast cancer. Cancer immunology research, 11(5), 687-702.
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2

Gene Expression Analysis from Ear Tissue

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For analysis of gene expression, ears were processed as described above and single cell suspension immediately placed in RNA lysis buffer (Qiagen). Homogenates were then passed through Qiashredder columns, and RNA was purified using RNAeasy mini kit (Qiagen), according to the manufacturer’s protocol. Reverse transcription was performed using Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen Life Technologies). Real-time PCR was performed on an ABI Prism 7900 sequence detection system (Applied Biosystems). PCR primer probe sets were pre-developed gene expression assays designed by Applied Biosystems, and the quantities of products were determined by the comparative threshold cycle method using the equation 2−ΔΔCt (where Ct represents cycle threshold) to determine the fold increase in product. Each gene of interest was normalized to the 18s rRNA endogenous control.
Charmoy M., Hurrell B.P., Romano A., Lee S.H., Ribeiro-Gomes F., Riteau N., Mayer-Barber K., Tacchini-Cottier F, & Sacks D.L. (2016). The Nlrp3 inflammasome, IL-1β, and neutrophil recruitment are required for susceptibility to a non-healing strain of Leishmania major in C57BL/6 mice. European journal of immunology, 46(4), 897-911.
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3

nCounter and HTS Assays for Toxicogenomics

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The nCounter assay was performed with 100 ng of RNA that had previously been pooled and used in the microarray analysis. Methodological details of the nCounter experiments have been published previously (26 (link)). In brief, optimized sequences for genes in the TGx-DDI panel were custom-designed and manufactured by NanoString. The CodeSet included the TGx-DDI gene set and eight housekeeping genes—G6PD, GUSB, HPRT1, LDHA, NONO, PGK1, PPIH, and TFRC—selected based on stability and detectable expression levels. The protocol followed standard nCounter instructions (26 (link)). Barcodes were counted for each target, and the data were exported. The counts of each target were analyzed using nSolver Analysis version 3.0 for quality control and normalization. Normalized data were subjected to further analysis.
To develop the HTS assay, 5 × 104 cells per well were seeded in a 96-well plate on the day before the treatment. Cells were treated with bleomycin and its corresponding vehicle control (H2O) for 4 h, rinsed to remove the drug, and then either lysed in RNA lysis buffer (products from Qiagen, Ambion, and Promega were tested and performed comparably) at different concentrations or pelleted for RNA isolation. This treatment was performed in triplicate, after which bioinformatics analyses were conducted as described above.
Li H.H., Chen R., Hyduke D.R., Williams A., Frötschl R., Ellinger-Ziegelbauer H., O’Lone R., Yauk C.L., Aubrecht J, & Fornace AJ J.r. (2017). Development and validation of a high-throughput transcriptomic biomarker to address 21st century genetic toxicology needs. Proceedings of the National Academy of Sciences of the United States of America, 114(51), E10881-E10889.
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4

Primary Hepatocyte Isolation and Culture

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Human primary hepatocytes were obtained from the Liver Tissue Cell Distribution System at the University of Pittsburgh (Pittsburgh, Pennsylvania, USA). Primary mouse hepatocytes were isolated from 10-week-old WT C57BL/6J mice, as described previously (32 (link), 81 (link)). All cells were cultured in DMEM containing 10% FBS and then transfected with siRNAs described in the figure legends. The cells were then cultured in serum-free medium until they were harvested into RIPA buffer (Thermo Fisher Scientific, catalog 89900) with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, catalog 78444) for immunoblotting or into RNA lysis buffer (QIAGEN, catalog 79216) for mRNA quantification. Culture media were collected, snap-frozen in liquid nitrogen, and stored at –80°C until processing.
Zheng Z., Nakamura K., Gershbaum S., Wang X., Thomas S., Bessler M., Schrope B., Krikhely A., Liu R.M., Ozcan L., López J.A, & Tabas I. (2020). Interacting hepatic PAI-1/tPA gene regulatory pathways influence impaired fibrinolysis severity in obesity. The Journal of Clinical Investigation, 130(8), 4348-4359.
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5

FACS Sorting of Adipocytes

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FACS sorting of adipocytes according to their GFP expression was described previously (56 (link)). Briefly, cell sorting of adipocytes was performed using an Aria III high-speed sorter (BD Bioscience). A nozzle of 130 mm diameter, sheath pressure of 10 psi and a standard 4-way purity mask as described in the sorter manual was used during all sorts. Transgenic Ucp1-eGFP mice, expressing the eGFP protein under the control of Ucp1 promoter, were cold acclimated at 8°C for 7 days and the mature adipocyte fractions from iBAT and iWAT were separated by FACS using eGFP. The adipocyte population was first defined in the forward and side scatter by size and internal complexity characteristics. The GFP+ population was defined in the respective gate and the mature brown adipocytes were isolated from the mature fraction of iBAT. The same strategy was applied for the mature adipocyte fraction of iWAT to isolate the GFP+ population, which constitutes brite adipocytes, and the adjacent GFP− population that constitutes white adipocytes. For each sample, 3 or 6 same gender mice were pooled per sample and 500–3,000 cells were collected directly in RNA Lysis Buffer (Qiagen) and kept on ice until frozen and stored at −80°C.
Müller S., Perdikari A., Dapito D.H., Sun W., Wollscheid B., Balaz M, & Wolfrum C. (2020). ESRRG and PERM1 Govern Mitochondrial Conversion in Brite/Beige Adipocyte Formation. Frontiers in Endocrinology, 11, 387.
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6

Senescence Profiling of Tumor-Infiltrating T Cells

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Peripheral blood samples (10 mL) were obtained from patients no more than two weeks prior to the start of immunotherapy. Most were obtained on the same day as therapeutic initiation (93.5%). Patient and control PBTLs were isolated from whole blood collected on the same day using negative selection and the EasySep Direct Human T cell Isolation Kit (StemCell Technologies #19661). The purity of control PBTLs was determined by flow cytometry using anti-CD3 antibody as described20 . Sample purity ranged from 61–99% (average = 96%; median = 97%). Purified PBTLs were suspended in RNA lysis buffer (Qiagen), flash frozen and stored at −80°C pending RNA extraction.
RNA was isolated from snap-frozen PBTL lysates using the Qiagen RNeasy mini kit. Samples with high quality RNA (RIN > 6.6) were converted to cDNA using SuperScript VILO Master Mix (Life Technologies). A subset of OSU_Senescence targets: CD244, CD274, CD276, CDKN2A (p14ARF), CDKN2A (p16INK4a), IL6 and IL17A, were then pre-amplified using custom Nanostring primers and Taqman PreAmp Master Mix. The resulting samples were subjected to standard nCounter analysis using a custom Nanostring CodeSet, OSU_Senescence (Supp. Table S2). Target counts were normalized to five internal controls (GUSB, HPRT1, PGK1, UBC, and YWAZ) using nSolver software. The resulting data were log2-transformed.
Galloway J.E., Holderbaum A.M., Arya N., Zhang S., Bodnar M.S., Norman R., Carson W.E., Yu L., Kendra K.L, & Burd C.E. (2020). Impact of age-related T cell dynamics on the identification of biomarkers predictive of immunotherapy discontinuation: a prospective cohort study. Aging and cancer, 1(1-4), 58-70.
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7

Investigating TLR2 and TLR5 Activation

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Agonists to TLR2 – a heat-killed preparation of Listeria monocytogenes (HKLM), and TLR5 – flagellin from Salmonella typhimurium, were purchased from InvivoGen. These agonists were used at working concentrations recommended by the manufacturer (HKLM - 108 cells/mL, flagellin - 250 ng/mL). Twenty four hours prior to agonist exposure, stratified HCLE-NT, HCLE-scrMUC1, HCLE-shMUC1, HCLE-scrMUC16, and HCLE-shMUC16 cells were switched to antibiotic- and EGF-free K-SFM. Cells were then exposed to the agonists that were pre-diluted in antibiotic- and EGF-free K-SFM for either 4 h (RNA analyses) or 12 h (protein analysis). In ex vivo experiments, eyes of WT and Muc1-/- mice were enucleated and independently incubated in antibiotic- and EGF-free K-SFM containing a cocktail of the TLR2 and TLR5 agonists at working concentrations described above. Following a 4 h exposure to the agonists, the corneal epithelium was carefully debrided using a blade attached to a micro blade holder. The debrided epithelium from four eyes of each strain was pooled to constitute one sample (total n=3 for WT and n=6 for Muc1-/-, each n=4 eyes). Pooled samples were collected in RNA lysis buffer (Qiagen) and stored at -80°C until further use.
Menon B.B., Kaiser-Marko C., Spurr-Michaud S., Tisdale A.S, & Gipson I.K. (2015). Suppression of Toll-like Receptor-Mediated Innate Immune Responses at the Ocular Surface by the Membrane-associated Mucins MUC1 and MUC16. Mucosal immunology, 8(5), 1000-1008.
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8

Brain Single-cell Isolation in Mice

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For brain isolation, mice were anesthetized with 1.5% isoflurane (Abbott, Germany) and transcardially perfused with PBS and 4% paraformaldehyde (PFA). Mouse brain specimens were macrodissected and dissociated using the Neural Tissue dissociation kit (NTDK, Miltenyi) and a single-cell suspension generated using the GentleMACS dissociator. Tissue suspensions were filtered through a 70-μM mesh filter and underwent red blood cell lysis (Pharm-Lyse BD) and myelin removal with Myelin Removal Beads (Miltenyi). Single-cell suspensions were blocked by incubation for 15 min at 4 °C in 2% rat serum and then stained with cell-surface antibodies and fixable viability dye for 30 min at 4 °C. Cells were subsequently washed twice with FACS buffer and sorted in PBS with 5% FCS using BD FACSAria II (BD Biosciences, USA) running with FACSDiva Software (BD Biosciences). Cells were then centrifuged and resuspended in RNA lysis buffer (Qiagen) before snap freezing in liquid nitrogen and storage at –80 °C. Data were analyzed with FlowJo X 10.0.7 software (FlowJo, Ashland, OR, USA).
Arseni L., Sharma R., Mack N., Nagalla D., Ohl S., Hielscher T., Singhal M., Pilz R., Augustin H., Sandhoff R., Herold-Mende C., Tews B., Lichter P, & Seiffert M. (2023). Sphingosine-1-Phosphate Recruits Macrophages and Microglia and Induces a Pro-Tumorigenic Phenotype That Favors Glioma Progression. Cancers, 15(2), 479.
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9

Single-cell RNA-seq of Mouse Cells

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20,000–35,000 cells per each sample were sorted directly into RNA lysis buffer (Qiagen, 79216) supplemented with 2 M dithiothreitol. RNA was isolated using RNAeasy plus micro kit (Qiagen, 74034). Briefly, homogenized lysate was transferred to a gDNA eliminator spin column placed in a 2 mL collection tube (supplied by the manufacturer). The following steps were conducted according to the instructions provided by the manufacturer. For all RNA samples, RNA integrity number (RIN) was ≥8.5. After RNA isolation, 1 ng of total RNA was used to generate full length cDNA using Clontech’s SMART-Seq v4 Ultra Low Input RNA Kit (634888). cDNA was then used to prepare libraries using Illumina Nextera XT DNA sample preparation kit (FC-131–1024). Libraries with unique barcodes were pooled at equal molar ratios and sequenced on Illumina NextSeq 500 sequencer to generate 150 bp single reads, following manufacturer’s protocol.
The reads were aligned using the STAR version 2.3.0 software that permits unique alignments to Mouse Ensembl genes. Differential expression was determined using edgeR software with default settings. Expression is given in Counts per million (CPM).
Ledo J.H., Liebmann T., Zhang R., Chang J.C., Azevedo E.P., Wong E., Silva H.M., Troyanskaya O.G., Bustos V, & Greengard P. (2020). Presenilin 1 phosphorylation regulates amyloid-β degradation by microglia. Molecular Psychiatry, 26(10), 5620-5635.
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10

Purification of Microglia from CK-p25 Mice

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2 week induced CK-p25 mice and age-matched controls were perfused with 50 mls PBS to wash away blood and minimize macrophage contamination in the brains. Hippocampal tissue was harvested immediately after perfusion and a single-cell suspension was prepared as described previously56 (link). Fluorescence-activated cell sorting (FACS) was then used to purify CD11b+ CD45low microglia cells using APC conjugated CD11b mouse clone M1/70.15.11.5 (Miltenyi Biotec, #130-098-088) and PE conjugated CD45 antibody (BD Pharmingen, #553081). Cells were collected directly into RNA lysis buffer (Qiagen, #74104).
Gjoneska E., Pfenning A.R., Mathys H., Quon G., Kundaje A., Tsai L.H, & Kellis M. (2015). Conserved epigenomic signals in mice and humans reveal immune basis of Alzheimer’s disease. Nature, 518(7539), 365-369.
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