Rneasy plus isolation kit

Manufactured by Qiagen

Sourced in United States, Canada

The RNeasy Plus Isolation Kit is a tool designed for the efficient extraction and purification of total RNA from a variety of biological samples. It utilizes a silica-membrane-based technology to capture and purify RNA, ensuring high-quality results for downstream applications.

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5 protocols using rneasy plus isolation kit

RNA-seq of U937 cells and HMECs

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For U937 cells, total RNA was isolated from 1 million cells using the RNeasy Plus Isolation Kit (Qiagen) following the manufacturer instructions. The library was prepared using the TruSeq mRNA kit (Illumina) according to the standard protocol. Sequencing was performed on HiSeq 4000 using 100-bp paired-end reads, and 50 million reads were obtained per sample. For HMECs, total RNA was isolated from ∼1 million cells using the RNeasy Plus Isolation Kit (Qiagen) following the manufacturer instructions. RNA-seq was performed on two replicates of cells treated with no siRNA, two replicates treated with control siRNA #1, two replicates with control siRNA #2 and three replicates each of cells treated with siRNA STAG2 #2 and siRNA STAG2 #3. The library was prepared using the NEBnext Ultra II Directional RNA Library kit according to the standard protocol from 1 μg isolated RNA. Barcoded libraries were pooled at molar ratios, and 86-bp single-end sequencing was performed using Ilumina NextSeq 500.

Oreskovic E., Wheeler E.C., Mengwasser K.E., Fujimura E., Martin T.D., Tothova Z, & Elledge S.J. (2022). Genetic analysis of cancer drivers reveals cohesin and CTCF as suppressors of PD-L1. Proceedings of the National Academy of Sciences of the United States of America, 119(7), e2120540119.

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Quantifying Viral Gene Expression in miRNA-Transfected Cells

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293FT cells were either left untransfected or were transfected with miR-124/143 precursors (Ambion: AM17100/PM10691/MIMAT0000422, AM17100/PM10883/MIMAT0000435) or with scrambled miR (Thermo Fisher Scientific AM17110) using Lipofectamine RNAiMAX Transfection Reagent, followed by infection with VG2025 at MOI = 1. Cells were harvested at 6 h post infection and RNA was extracted using the RNeasy Plus Isolation Kit (QIAGEN) in accordance with the manufacturer’s instructions. RNA concentrations were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific). The High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) was used for reverse transcription and cDNA synthesis from quantified mRNA. qRT-PCR was performed to measure ICP27 and ICP34.5 expression and copy numbers using the TaqMan FastAdvanced Master Mix and QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). Plasmid DNA containing the measured target was used as standards for the absolute quantification of viral gene copy numbers.

Chouljenko D.V., Murad Y.M., Lee I.F., Delwar Z., Ding J., Liu G., Liu X., Bu X., Sun Y., Samudio I, & Jia W.W. (2023). Targeting carcinoembryonic antigen-expressing tumors using a novel transcriptional and translational dual-regulated oncolytic herpes simplex virus type 1. Molecular Therapy Oncolytics, 28, 334-348.

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Profiling Host Immune Responses Post Viral Therapy

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Gene expression analysis was performed with real-time RT-PCR. A total of 5 × 10⁷ PFU of VG161 was intratumorally administered to mice bearing the CT26 tumors. Tumors were collected 24 h after the virus injection, and the mRNA was isolated using RNeasy Plus isolation kit (Qiagen, Frederick, MD, USA). RT-PCR amplification was performed using the RT² First Strand Kit. We used the RT² Profiler™ PCR Array Mouse Innate & Adaptive Immune Responses (PAMM-052Z) with 84 mouse genes involved in the host innate and adaptive immune response for the gene expression profiling, and RT² SYBRR Green qPCR Master Mix (all from Qiagen, Frederick, MD, USA) was used for setting up the qPCR reactions. Thermal cycling was performed using ABI-7000 (Applied Biosystems, Foster, CA, USA). Data analysis was performed, and a heat map was generated using online analysis tools available on the Qiagen data analysis center.

Chouljenko D.V., Ding J., Lee I.F., Murad Y.M., Bu X., Liu G., Delwar Z., Sun Y., Yu S., Samudio I., Zhao R, & Jia W.W. (2020). Induction of Durable Antitumor Response by a Novel Oncolytic Herpesvirus Expressing Multiple Immunomodulatory Transgenes. Biomedicines, 8(11), 484.

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Quantitative RT-PCR for Bmp1 Gene Expression

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Complete RNA was isolated using the RNeasy plus isolation kit (Qiagen Valencia, CA), and quality and quantity was confirmed by NanoDrop. cDNA was generated using iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA). qPCR for Bmp1 gene transcripts and β-actin were performed with iQ SYBR green Supermix (Bio-Rad) on a BioRad MyiQ PCR machine. The sequence and conditions for amplification of transcripts from the Bmp1 gene were as follows: forward primer 5’-GACTCACGGCGGACTCTAAG-3’; reverse primer, 5’-CACTGGTGGATGTCACCTTG-3’; 95°C, 3 minutes, 1 cycle; followed by 95°C for 30 sec; 53°C for 30 sec; 72°C for 1 minute; for 22 cycles; 72°C for 2 minutes, 1 cycle. The sequence and conditions for β-actin were as follows: forward primer, 5’-GCCAGGTCATCACTATTGG-3’; reverse primer, 5’-AGTAACAGTCCGCCTAGAAGC-3’; 94°C, 3 minutes, 1 cycle; followed by 94°C for 1 minute; 58°C for 1 minute 30 sec; 72°C for 1 minute 30 sec; for 18 cycles; 72°C for 2 minutes, 1 cycle.

Longmate W.M., Lyons S.P., DeFreest L., Van De Water L, & DiPersio C.M. (2017). Opposing roles of epidermal integrins α3β1 and α9β1 in regulation of mTLD/BMP-1-mediated laminin-γ2 processing during wound healing. The Journal of investigative dermatology, 138(2), 444-451.

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Quantifying Cardiac Gene Expression in Human Engineered Cardiac Tissues

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To determine gene expression of the tissues, total RNA was extracted from the hECTs using Trizol (Life Technologies) and the RNeasy plus isolation kit (Qiagen). RNA was reverse transcribed using oligo-dT primers with the Superscript II Synthesis Kit (Life Technologies). qPCR was completed with Fast SYBR Green Mastermix (Applied Biosystems) according to the manufacturer’s instructions. The primers used were cardiac troponin (cTnT), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2a). Gene expression was normalized against the housekeeping gene GAPDH using the ΔΔC_t method. The expression level of cardiac genes is reported relative to expression of cardiac-specific cTnT. A StepOne Plus Real-Time PCR System (Applied Biosystems) was used to perform the qPCR and analyzed with StepOne Software v2.2.2.

Cashman T.J., Josowitz R., Johnson B.V., Gelb B.D, & Costa K.D. (2016). Human Engineered Cardiac Tissues Created Using Induced Pluripotent Stem Cells Reveal Functional Characteristics of BRAF-Mediated Hypertrophic Cardiomyopathy. PLoS ONE, 11(1), e0146697.

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