Reliaprep rna tissue miniprep system

Manufactured by Promega

Sourced in United States, Germany, Japan, Switzerland

The ReliaPrep RNA Tissue Miniprep System is a lab equipment product designed for the isolation of total RNA from a variety of tissue samples. The system utilizes a simple and efficient spin column-based method to extract high-quality RNA for downstream applications such as gene expression analysis, RT-PCR, and other RNA-based experiments.

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ReliaPrep™ RNA Miniprep Systems (165 citations) ReliaPrep RNA Tissue Miniprep System kit (11 citations) RNA Tissue Miniprep System (5 citations) ReliaPrep™ Tissue Miniprep System (3 citations) ReliaPrep RNA tissues miniprep system (2 citations) ReliaPrep™ RNA Tissue Miniprep (2 citations) ReliaPrepTM RNA Tissue (2 citations) RNA Miniprep Systems (2 citations) Minipreps (2 citations)

155 protocols using reliaprep rna tissue miniprep system

Quantifying Poplar Gene Expression

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Frozen stems were scraped with a scalpel. Subsequently, the xylem material (which was removed from the stem piece with a scalpel) was ground to fine powder using a mortar/pestle and a Retsch MM300 mill (20 Hz, 5-mm bead). Total RNA was isolated using the ReliaPrep™ RNA Tissue Miniprep System (Promega). To eliminate genomic DNA contamination, on-column DNase digestion was performed (included in the ReliaPrep™ RNA Tissue Miniprep System (Promega)). RNAs from two plants were pooled to constitute a replicate. Total RNA (1 μg) was used as a template for the synthesis of cDNA using qScript® cDNA SuperMix (Quantabio). The transcript levels of AtCCR1 (AT1G15950) were determined with the Roche LightCycler 480 combined with the SYBR Green I Master Kit (Roche Diagnostics) in three technical repeats.
Poplar 18S RIBOSOMAL RNA (AF206999), POLYUBIQUITIN (BU879229), and LEAFY/FLORICAULA (Potri.015G106900) were used as reference genes. All primers used in this study are listed in Table S2. For the statistical analysis, normalized relative quantities were log10 transformed.

De Meester B., Vanholme R., de Vries L., Wouters M., Van Doorsselaere J, & Boerjan W. (2021). Vessel- and ray-specific monolignol biosynthesis as an approach to engineer fiber-hypolignification and enhanced saccharification in poplar. The Plant journal : for cell and molecular biology, 108(3).

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Pancreatic RNA Extraction and qPCR Analysis

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Pancreatic tissue was immediately placed in RNAlater^TM solution (Invitrogen, no. AM7020) after snap-freezing in liquid nitrogen. Total RNA was first extracted with TRIzol reagent (Takara, no. 9109) followed by a ReliaPrep^TM RNA Tissue Miniprep system according to the manufacturer’s instructions (Promega, Madison, WI, USA). RNA integrity was observed using agarose gel electrophoresis. Then, cDNA was synthesized using ReverTra Ace^® qPCR RT Master Mix according to the manufacturer’s instructions (Toyobo, Osaka, Japan). mRNA expression was analyzed by qPCR on a Step-One RT-PCR system (Applied Biosystems) using SYBR green incorporation. The raw data were analyzed by the 2^−ΔΔCT method and normalized to Hprt expression. The primer sequences used in this study are shown in Supplementary Table 2.

Boonchan M., Arimochi H., Otsuka K., Kobayashi T., Uehara H., Jaroonwitchawan T., Sasaki Y., Tsukumo S.I, & Yasutomo K. (2021). Necroptosis protects against exacerbation of acute pancreatitis. Cell Death & Disease, 12(6), 601.

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RNA-seq analysis of Drosophila CNS

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L3 larvae (n=45) were collected and quickly washed in water to remove food and yeast. Larvae were placed in ice-cold PBS in a depression well. Larval CNSs were fine dissected and transferred into microcentrifuge tubes containing 250 μl of an ice-cold 1:1 solution of RNAse-free PBS and RNALater (Invitrogen, AM7020). CNS samples were centrifuged at 382 g for 5 min at 4°C, and the supernatant was removed. Then, 500 μl fresh RNALater solution was added, and samples were stored at −80°C. Once CNS collection for three biological replicates per genotype had been performed, RNA extraction was carried out according to the manufacturer's protocol (Promega ReliaPrepTM RNA Tissue Miniprep System, Z6111). RNA concentration was measured using NanoDrop OneC (Thermo Fisher Scientific), and RNA integrity was checked by agarose gel electrophoresis. Four to six micrograms of total RNA per biological replicate were sequenced (Novogene Co. Ltd). Samples were assessed for quality with an Agilent 2100 Bioanalyzer system, and paired-end sequencing was performed on an Illumina platform. Over 40 million reads/genotype were generated and mapped to the genome at a rate of 94-96%. Drosophila melanogaster (ensemble bdgp6_gca_000001215_4 genome assembly) was used. HISAT2 algorithm for alignment and DESeq2 R package (Anders and Huber, 2010 (link)) for differential gene expression were used.

Pfeifer K., Wolfstetter G., Anthonydhason V., Masudi T., Arefin B., Bemark M., Mendoza-Garcia P, & Palmer R.H. (2022). Patient-associated mutations in Drosophila Alk perturb neuronal differentiation and promote survival. Disease Models & Mechanisms, 15(8), dmm049591.

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RNA Isolation and RT-qPCR from Retina and Optic Nerve

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Retinal tissue was homogenized by trituration using a pipette (n = 5/group). The RNA isolation of the retina was carried out according to the manufacturer's introduction using the Gene Elute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA). For total RNA isolation of optic nerve tissue, the ReliaPrepTM RNA Tissue Miniprep System (Promega, Madison, WI, USA) was taken. Prior to isolation, optic nerve tissue was incubated in liquid nitrogen and then homogenized with a pestle (n = 5/group). An additional DNase digestion at RT for 15 min ensured that no genomic DNA contaminated RNA was obtained. The concentration and purity of the isolated RNA was determined photometrically using the BioSpectrometer® (Eppendorf, Hamburg, Germany). One microgram RNA and random hexamer primers were applied for reverse transcription using a cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). RT-qPCR experiments were done with SYBR Green I in a Light Cycler 96® (Roche Applied Science, Mannheim, Germany). For each primer pair (Table 4), efficiencies were determined by a dilution series of 5, 25, and 125 ng cDNA. Expression in retina and optic nerve tissue was normalized against the housekeeping genes β-actin (Actb) and 18S ribosomal RNA (Rn18S), respectively.

Wiemann S., Reinhard J., Reinehr S., Cibir Z., Joachim S.C, & Faissner A. (2020). Loss of the Extracellular Matrix Molecule Tenascin-C Leads to Absence of Reactive Gliosis and Promotes Anti-inflammatory Cytokine Expression in an Autoimmune Glaucoma Mouse Model. Frontiers in Immunology, 11, 566279.

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Retinal and Optic Nerve RNA Extraction

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For RNA isolation, retinae (n = 3–6/group) were prepared, stored in lysis buffer and snap frozen in liquid nitrogen 3, 7 and 14 days after immunization. Total retinal RNA was extracted using the Gene Elute Mammalian Total RNA Miniprep Kit according to the manufacturer's instructions. Optic nerve RNA (n = 3/group) was extracted using the ReliaPrepTM RNA Tissue Miniprep system (Promega, Madison, WI, USA) from snap frozen tissue 7 days after immunization. The quality and quantity of RNA were assessed by measurement of the ratio of absorbance values at 260 and 280 nm (BioSpectrometer^®; Eppendorf, Hamburg, Germany). Total RNA (1 μg) was used for reverse transcription using cDNA synthesis kit (Thermo Fisher Scientific).

Reinehr S., Reinhard J., Wiemann S., Stute G., Kuehn S., Woestmann J., Dick H.B., Faissner A, & Joachim S.C. (2016). Early remodelling of the extracellular matrix proteins tenascin‐C and phosphacan in retina and optic nerve of an experimental autoimmune glaucoma model. Journal of Cellular and Molecular Medicine, 20(11), 2122-2137.

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RNA Extraction and Reverse Transcription

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RNA was extracted using the ReliaPrepTM RNA Tissue Miniprep System (Promega^®) followed by DNase treatment according to the manufacturer’s instructions. The RNA quantification and purity were assured using a Nanodrop ND-100 spectrophotometer (Thermo-Fischer Scientific^®), and samples with 260/280 ratios between 1.8 and 2.2 were considered sufficiently pure. The samples’ integrity was also checked by electrophoresis on a 1% agarose gel and stained with SYBR Safe DNA gel stain (Thermo-Fischer Scientific^®). Approximately 1 µg of each RNA sample was reverse transcribed with random hexamers by SuperScript III Reverse Transcriptase (Thermo-Fischer Scientific^®). The samples were tested for the absence of genomic DNA with UPD-apiose/UDP-xylose synthase (AXS) primers (Forward: 5’-GCATCCAGTTCCACCGTCTC-3’; Reverse: 5’-GCAGGGCGTTTCATCTTCTTT-3’) as described by Pagliuso et al. (2022) (link).

Pagliuso D., Pedro de Jesus Pereira J., Ulrich J.C., Barbosa Cotrim M.E., Buckeridge M.S, & Grandis A. (2023). Carbon allocation of Spirodela polyrhiza under boron toxicity. Frontiers in Plant Science, 14, 1208888.

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Quantitative Real-Time PCR Analysis

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Total RNA from primary cells and bone tissue was extracted using ReliaPrep^TM RNA Tissue Miniprep System (Promega, Madison, WI, USA) and TRIzol reagent (Invitrogen, Darmstadt, Germany), respectively, following the manufacturer’s protocol and quantified using a Nanodrop spectrophotometer (Peqlab, Erlangen, Germany).
Five hundred nanograms of RNA were reverse transcribed using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) followed by GoTaq® qPCR Master Mix-based quantitative real-time PCR (Promega, Madison, WI, USA) according to established protocols (ABI7500 Fast, Applied Biosystems, Carlsbad, CA, USA). Primer sequences for mice are listed in supplemental Table S1. PCR conditions were: 50 °C for 5 min and 95 °C for 10 min followed by 40 cycles with 95 °C for 15 s and 60 °C for 1 min. Melting curves were evaluated using the following scheme: 95 °C for 15 s, 60 °C for 1 min and 95 °C for 30 s. Results were calculated based on the ∆∆CT method and are represented as x-fold increase normalized to β-actin and GAPDH mRNA levels.

Lademann F., Rijntjes E., Köhrle J., Tsourdi E., Hofbauer L.C, & Rauner M. (2024). Hyperthyroidism-driven bone loss depends on BMP receptor Bmpr1a expression in osteoblasts. Communications Biology, 7, 548.

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Quantitative PCR Analysis of Cardiac Tissues

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Human or mouse cardiac tissues, isolated human cardiomyocytes or iPSC-cardiomyocytes were snap-frozen in liquid nitrogen and stored at −80 °C. RNA was isolated by use of the ReliaPrep^TM RNA Tissue Miniprep System (Promega). About 200 ng RNA was reverse transcribed into cDNA using standard protocols (QuantiNova Reverse Transcription Kit (QIAGEN) for mouse tissue and iScript cDNA Synthesis Kit (Bio-Rad) for human samples). For qPCR, 10 µL SYBR Green PCR Master Mix (Bio-Rad), 7 µL nuclease-free water, 1 µL forward and 1 µl reverse Primer, and 1 µL of cDNA were mixed. Q-PCR was carried out using the CFX ConnectTM Real-Time System (Bio-Rad). Forty cycles of 15 s at 95 °C followed by 1 min of 60 °C were used and fluorescence was measured after each cycle. After 40 cycles melt curve analysis was performed to ensure the specificity of the products. Thresholds cycles were evaluated and normalized to housekeeping genes and controls. A list of all primers used is presented in Supplementary Table 4.

Bengel P., Dybkova N., Tirilomis P., Ahmad S., Hartmann N., A. Mohamed B., Krekeler M.C., Maurer W., Pabel S., Trum M., Mustroph J., Gummert J., Milting H., Wagner S., Ljubojevic-Holzer S., Toischer K., Maier L.S., Hasenfuss G., Streckfuss-Bömeke K, & Sossalla S. (2021). Detrimental proarrhythmogenic interaction of Ca2+/calmodulin-dependent protein kinase II and NaV1.8 in heart failure. Nature Communications, 12, 6586.

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RNA-seq Analysis of Drosophila Embryos

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Embryos were collected at 25°C (5-16 h after egg laying) and dechorionated in 2% sodium hypochlorite solution for 4 min. After subsequent washing with embryo wash (0.8% NaCl and 0.05% Triton X-100) and H₂O, embryos were stored at −80°C. One embryo collection for three biological replicates per genotype was obtained. RNA-extraction was carried out according to the manufacturer's protocol (Promega ReliaPrepTM RNA Tissue Miniprep System, REF-Z6111). Total RNA was measured using NanoDrop OneC (Thermo Scientific) for its concentration and RNA integrity was checked using gel electrophoresis. Then 9-15 µg of total RNA/biological replicate was shipped to Novogene for sequencing. Prior to making the library, samples were reassessed for quality with the Agilent 2100 Bioanalyzer system. Sequencing was performed on an Illumina platform and paired-end reads were produced. Over 40 million reads/genotype were generated and mapped to the genome at a rate of over 96%. Drosophila melanogaster (ensemble bdgp6_gca_000001215_4 genome assembly) was used. HISAT2 algorithm for alignment and DESeq2 R package (Anders and Huber, 2010 (link)) for differential gene expression was used. Subsequent analyses were performed with GraphPad Prism 9. Fold change ≥1.5 and ≤−1.5 (log2FC≥0.59 and ≤−0.59) for up- and downregulated genes, respectively, and P_adj≤0.05 were used for statistical significance.

Mendoza-Garcia P., Basu S., Sukumar S.K., Arefin B., Wolfstetter G., Anthonydhason V., Molander L., Uçkun E., Lindehell H., Lebrero-Fernandez C., Larsson J., Larsson E., Bemark M, & Palmer R.H. (2021). DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as an Alk target in the Drosophila visceral mesoderm. Development (Cambridge, England), 148(23), dev199465.

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Aortic Gene Expression Analysis

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The mRNA expression levels of VCAM-1, ICAM-1, and MCP1 were determined in the aortas by RT-PCR. About 2 mg of frozen aortic tissue was homogenized, and total RNA was isolated using the ReliaPrep^TM RNA Tissue Miniprep System (Promega, Madison, WI, USA) according to the protocol provided by the manufacturer. The concentration of total RNA was quantified by spectrophotometry (ND-1000; NanoDrop Technologies, Wilmington, DE, USA). The A260/A280 ratio of the spectrograms was confirmed between 1.8–2.0, and the A260/A230 ratio was always greater than 1.5. RNA was reverse transcribed to single-stranded cDNA using the SuperScript III First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was subjected to quantitative PCR analysis with FastStart Universal Probe Master Mix (Roche, Basel, Switzerland) using primers for VCAM-1, ICAM-1, and MCP-1 using an ABI 7300 Real-Time PCR system (Thermo Fisher Scientific), as described previously [58 (link)]. The primers and probes were designed by the Universal ProbeLibrary Assay Design Center (Roche, Basel, Switzerland). The relative level of mRNA expression of each gene was expressed relative to that of β-actin.

Yokoyama Y., Mise N., Suzuki Y., Tada-Oikawa S., Izuoka K., Zhang L., Zong C., Takai A., Yamada Y, & Ichihara S. (2018). MicroRNAs as Potential Mediators for Cigarette Smoking Induced Atherosclerosis. International Journal of Molecular Sciences, 19(4), 1097.

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