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Mirneasy mini column

Manufactured by Qiagen
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The MiRNeasy mini columns are used for the purification of total RNA, including small RNAs such as miRNA, from various sample types. The columns are designed to efficiently capture and purify RNA molecules of all sizes, allowing for the comprehensive analysis of the entire RNA content of a sample.

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Lab products found in correlation

Qiazol, by Qiagen (3 mentions) Viia 7 real time pcr system, by Thermo Fisher Scientific (2 mentions) Truseq stranded total rna library prep human mouse rat kit, by Illumina (2 mentions) Novaseq, by Illumina (2 mentions) Aria 2, by BD (2 mentions) Qiashredder, by Qiagen (2 mentions) Rnase free dnase, by Qiagen (2 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions) Nanodrop 2000, by Thermo Fisher Scientific (2 mentions) Wt ovation pico rna amplification system, by Tecan (1 mentions) Fl ovation cdna biotin module v2, by Tecan (1 mentions) Maxima reverse transcriptase, by Thermo Fisher Scientific (1 mentions) Quantifast sybr green pcr kit, by Qiagen (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Taqman master mix, by Thermo Fisher Scientific (1 mentions) Transcriptor first strand cdna synthesis kit, by Roche (1 mentions) Rnaseout, by Thermo Fisher Scientific (1 mentions) Qiazol lysis reagent, by Qiagen (1 mentions) Taqman fast universal pcr master mix, by Thermo Fisher Scientific (1 mentions) Quantstudio 3, by Thermo Fisher Scientific (1 mentions)

9 protocols using mirneasy mini column

1

Transcriptome analysis of Drosophila hearts

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Drosophila hearts from 1‐ and 5‐week‐old yw wildtype or yw x GMH5 flies (n = 30) were isolated (Fink et al., 2009), removed and homogenized in TRIzol (Invitrogen Life Sciences). Total RNA was precipitated and purified by miRNeasy mini‐column (Qiagen) according to the manufacturer's instructions. The recovered RNA was subjected to first and second strand cDNA synthesis, amplification, and purification using the WT‐ovation Pico RNA Amplification System (NuGEN), Agencourt RNAClean purification beads (Beckman Coulter), and DNA Clean and Concentrator‐25s columns (Zymo Research). cDNA was fragmented and labeled using the FL‐ovation cDNA Biotin Module V2 (NuGEN).
Cannon L., Zambon A.C., Cammarato A., Zhang Z., Vogler G., Munoz M., Taylor E., Cartry J., Bernstein S.I., Melov S, & Bodmer R. (2017). Expression patterns of cardiac aging in Drosophila. Aging Cell, 16(1), 82-92.
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2

Quantifying Transcriptional Changes in C. elegans

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Hypochlorite-synchronized L1 stage wild type or mutant animals were grown for 36 hours post-hatching at 20°C on standard NGM plates with OP-50 E. coli. Animals were washed off the plates using isotonic M9 solution. RNA was extracted using TRIzol (Invitrogen) followed by miRNeasy mini column (Qiagen). cDNA was generated from 1-2 mg of total RNA using random hexamers and Maxima Reverse Transcriptase (Thermo Fisher Scientific). Quantitative PCR was performed on the ViiA 7 Real-Time PCR System (Life Technologies) using the QuantiFast SYBR Green PCR Kit (Qiagen). Thermocycling was done for 40 cycles in a two-step cycling in accordance with the manufacturer’s instructions and each PCR reaction was performed in triplicate. Changes in mRNA expression were quantified using act-3 mRNA as a reference.
Sepulveda G.P., Gushchanskaia E.S., Mora-Martin A., Esse R., Nikorich I., Ceballos A., Kwan J., Blum B.C., Dholiya P., Emili A., Perissi V., Cardamone M.D, & Grishok A. (2024). DOT1L stimulates MYC/Mondo transcription factor activity by promoting its degradation cycle on chromatin. bioRxiv.
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3

CRISPR-Seq Profiling in K562 Cells

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K562s expressing dCas9-BFP-KRAB were spinfected in biological duplicate with lentivirus expressing GFP and an sgRNA. Two days after spinfection, the cells were sorted for GFP+ on a BD ARIA II. Sort purity was generally >95%. After the sort, cells were maintained in media supplemented with 4 ug/ml puromycin for four days and then recovered for two days. Cells were counted, collected by centrifugation, and harvested by vigorous vortexing in Qiazol (Qiagen 79306).
Total RNA was extracted with miRNeasy Mini columns (Qiagen 217004) according to the manufacturer’s instructions and sequencing libraries were prepared with TruSeq Stranded Total RNA Library Prep Human/Mouse/Rat kits (Illumina 20020596) according to the manufacturer’s instructions. Libraries were sequenced 2x150 on a NovaSeq (Illumina).
Bulk RNA-seq data were aligned and quantified using STAR (version 2.7.9a) with parameters alignEndsType=Local and outFilter-MultimapNmax=20. For comparison of gene-level expression profiles, gene counts were corrected for sequencing depth (reads per million), and the log2 fold-change for each gene was calculated relative to within-replicate non-targeting control expression. The two replicates for each genetic perturbation were averaged in order to produce the final data.
Replogle J.M., Saunders R.A., Pogson A.N., Hussmann J.A., Lenail A., Guna A., Mascibroda L., Wagner E.J., Adelman K., Lithwick-Yanai G., Iremadze N., Oberstrass F., Lipson D., Bonnar J.L., Jost M., Norman T.M, & Weissman J.S. (2022). Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq. Cell, 185(14), 2559-2575.e28.
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4

Real-time PCR gene expression assay

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For real-time PCR, total RNA was extracted from cells with the miRNeasy mini columns (Qiagen) and 1 µg RNA was reverse-transcribed using the Transcriptor First Strand c-DNA Synthesis kit (Roche, Indianapolis, IN, USA). Probes for human SNAI2 (Hs_00950344_m1), LRIG1 (Hs_00394267_m1), EGFR (Hs_01076090_m1), and GAPDH (Hs_02758991_g1) were purchased from Applied Biosystems (Foster City, USA). Real-time PCR was carried out in triplicate using TaqMan Master Mix (Applied Biosystems) and samples were analyzed through the ViiA 7 Real-Time PCR System (Applied Biosystems). All transcript levels were normalized to that of GAPDH.
Majorini M.T., Manenti G., Mano M., De Cecco L., Conti A., Pinciroli P., Fontanella E., Tagliabue E., Chiodoni C., Colombo M.P., Delia D, & Lecis D. (2018). cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells. Cell Death and Differentiation, 25(12), 2147-2164.
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5

Robust RNA Extraction from Frozen Tissue

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RNA was extracted from the tissues denoted using a liquid nitrogen cooled mortar and pestle. Ground, frozen tissue was transferred to Qiazol lysis reagent (Qiagen; Valencia, CA, United States) and further homogenized using QIAshredders (Qiagen; Valencia, CA, United States). RNA was then isolated using the miRNeasy mini columns as described by the manufacturers’ protocol (Qiagen; Valencia, CA, United States). Following elution from the miRNeasy column, RNA was treated with RNase-free DNase (Qiagen, Valencia, CA, United States) for 25 min at room temperature, ethanol precipitated and resuspended in nuclease-free water supplemented with 1.25% RNaseOUT (Life Technologies; Carlsbad, CA, United States).
Kramer M.C., Kim H.J., Palos K.R., Garcia B.A., Lyons E., Beilstein M.A., Nelson A.D, & Gregory B.D. (2022). A Conserved Long Intergenic Non-coding RNA Containing snoRNA Sequences, lncCOBRA1, Affects Arabidopsis Germination and Development. Frontiers in Plant Science, 13, 906603.
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6

CRISPR-Seq Profiling in K562 Cells

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K562s expressing dCas9-BFP-KRAB were spinfected in biological duplicate with lentivirus expressing GFP and an sgRNA. Two days after spinfection, the cells were sorted for GFP+ on a BD ARIA II. Sort purity was generally >95%. After the sort, cells were maintained in media supplemented with 4 ug/ml puromycin for four days and then recovered for two days. Cells were counted, collected by centrifugation, and harvested by vigorous vortexing in Qiazol (Qiagen 79306).
Total RNA was extracted with miRNeasy Mini columns (Qiagen 217004) according to the manufacturer’s instructions and sequencing libraries were prepared with TruSeq Stranded Total RNA Library Prep Human/Mouse/Rat kits (Illumina 20020596) according to the manufacturer’s instructions. Libraries were sequenced 2x150 on a NovaSeq (Illumina).
Bulk RNA-seq data were aligned and quantified using STAR (version 2.7.9a) with parameters alignEndsType=Local and outFilter-MultimapNmax=20. For comparison of gene-level expression profiles, gene counts were corrected for sequencing depth (reads per million), and the log2 fold-change for each gene was calculated relative to within-replicate non-targeting control expression. The two replicates for each genetic perturbation were averaged in order to produce the final data.
Replogle J.M., Saunders R.A., Pogson A.N., Hussmann J.A., Lenail A., Guna A., Mascibroda L., Wagner E.J., Adelman K., Lithwick-Yanai G., Iremadze N., Oberstrass F., Lipson D., Bonnar J.L., Jost M., Norman T.M, & Weissman J.S. (2022). Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq. Cell, 185(14), 2559-2575.e28.
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7

Quantitative Real-Time PCR Protocol

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To perform real-time PCR, total RNA was extracted from cells with the miRNeasy Mini Columns (Qiagen) and 1 mg RNA was reverse-transcribed by using the High Capacity cDNA Reverse Transcription Kit (#4368814, Thermo Fisher Scientific). Probes for human KRT8 (Hs_01595539_g1), ESR1 (Hs_01046816_m1), PGR (Hs_01556702_m1), GAPDH (Hs_02786624_g1); mouse Cpa3 (Mm00483940_m1), Erbb2 (Rn01461185_m1), Ms4a2 (Mm00442778_m1) and Gapdh (Mm99999915_g1) were purchased from Thermo Fisher Scientific. Probes for human BCL2 (Hs. PT.56a.2905156) and for mouse Krt5 (Mm.PT.58.41573083), Esr1 (Mm.PT.58.8025728), Pgr (Mm.PT.58.10254276) were purchased from IDT. Real-time PCR was carried out with TaqMan Fast universal PCR Master Mix (#4352042, Thermo Fisher Scientific) and samples were analyzed by QuantStudio 3 (Thermo Fisher Scientific). All transcript levels were quantified by using the DC t method and normalized to GAPDH expression.
Majorini M.T., Cancila V., Rigoni A., Botti L., Dugo M., Triulzi T., De Cecco L., Fontanella E., Jachetti E., Tagliabue E., Chiodoni C., Tripodo C., Colombo M.P, & Lecis D. (2020). Infiltrating Mast Cell-Mediated Stimulation of Estrogen Receptor Activity in Breast Cancer Cells Promotes the Luminal Phenotype. Cancer research, 80(11).
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8

RNA-seq protocol for PAM50 scores

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Cell lines were plated in 60 mm plates in regular media. When cells were ~ 70% confluent, cells were washed with PBS and lysed with 0.7 mL Qiazol (Qiagen, Germantown, MD) immediately. RNA was prepared using miRNeasy mini columns (Qiagen) and treated with RNase-free DNase. RNA concentration was measured using a Nanodrop 2000 (Thermo-Fisher), and integrity analyzed using an Agilent 2100 Bioanalyzer and the RNA 6000 Nano kit. Libraries were prepared using the Illumina TruSeq Stranded mRNA Library Prep kit and samples sequenced using the Illumina Novaseq System. Paired-end 150 nt reads were aligned to the human genome version GRCh38.p13 using STAR 2.6. The downstream expression analysis was done using Cufflinks 2.2.1. PAM50 scores were assigned from the expression values (TPM) using the R package genefu 2.18.1 [27 (link)].
Finlay-Schultz J., Jacobsen B.M., Riley D., Paul K.V., Turner S., Ferreira-Gonzalez A., Harrell J.C., Kabos P, & Sartorius C.A. (2020). New generation breast cancer cell lines developed from patient-derived xenografts. Breast Cancer Research : BCR, 22, 68.
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9

Arabidopsis Growth and RNA Extraction

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Plant growth took place in a chamber with a controlled cycle of 16 light hours and 8 dark hours at 22 °C. The ecotype of Arabidopsis thaliana was UPQ10:NTF/ACT2p:BirA Columbia-0. Salt concentrations were based on fresh weight decreases as previously determined32 (link).
Rosette leaves were crushed in liquid N2 and RNA was extracted using Qiazol lysis reagent. QIAshredders (QIAGEN) were used to homogenize samples and miRNeasy mini columns (QIAGEN) were used to extract RNA. Samples were treated with RNase-free DNase (QIAGEN) for 30 minutes at room temperature and then RNA was ethanol precipitated.
Janssen K.A., Xie Y., Kramer M.C., Gregory B.D, & Garcia B.A. (2022). Data Independent Acquisition for the Detection of Mononucleoside RNA Modifications by Mass Spectrometry. Journal of the American Society for Mass Spectrometry, 33(5), 885-893.
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