SBC3/SBC3-miR-1Zip, SBC5/SBC5-miR-1-Tet-ON (-DOX/ + DOX) cells were lysed using RNA lysis buffer provided with the mirVana RNA isolation kit (Cat#AM1561, Thermo Fischer Scientific). Total RNA was isolated by following the manufacturer’s protocol. Whole transcriptome analysis (RNA sequencing) was performed on SBC3/SBC3-miR-1Zip, SBC5/SBC5-miR-1-Tet-ON (-DOX/ + DOX) cells at Sequencing Core Facility, City of Hope Comprehensive Cancer Center and Beckman Research Institute. RNA quality was determined using Agilent Bioanalyzer (Agilent Technologies, Inc.) and RNA samples with integrity numbers (RINs) of 10 were used for further analyses. Library preparation, PCR amplification, size distribution, library quantification, and sample loading were performed as described previously [23 (link)]. Sequencing was performed on a HiSeq2500 sequencer (Illumina Inc., San Diego, CA) in rapid mode. A single read, 50 cycle, sequencing run and onboard clustering and V2 chemistry were used. Raw files for RNA-seq were submitted NCBI SRA (accession number PRJNA900568).
Mirvana rna isolation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada, Brazil, Germany, United Kingdom, Spain, Lithuania
The MirVana RNA isolation kit is a product designed for the isolation and purification of RNA from various sample types, including cells, tissues, and body fluids. The kit utilizes a guanidinium-based lysis and acid-phenol extraction method to efficiently capture and recover RNA molecules, including small RNAs such as microRNAs. The kit provides a simple and reliable procedure for the extraction of high-quality RNA suitable for downstream applications.
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Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (37 mentions)
Nanodrop, by Thermo Fisher Scientific (14 mentions)
Trizol reagent, by Thermo Fisher Scientific (13 mentions)
Nanodrop nd 2000, by Thermo Fisher Scientific (13 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (12 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (12 mentions)
2100 bioanalyzer, by Agilent Technologies (9 mentions)
Trizol, by Thermo Fisher Scientific (8 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (8 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (7 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (6 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (6 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (6 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Hiseq 2500, by Illumina (5 mentions)
Hybond n membrane, by GE Healthcare (5 mentions)
Genespring software, by Agilent Technologies (5 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Hiseq 2000, by Illumina (5 mentions)
307 protocols using mirvana rna isolation kit
1
Transcriptome Analysis of miR-1 Regulation
2
RNA-seq transcriptome analysis of NRDE2 knockdown
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Triplicate RNA samples from si-Control and si-NRDE2-2-treated MDA-MB-231 cells were collected 48 h post-transfection using the mirVana RNA isolation kit (Thermo Fisher). Libraries were synthesized using Illumina TruSeq Stranded mRNA sample preparation kits from 500 ng of purified total RNA according to the manufacturer's protocol. The final dsDNA libraries were quantified by Qubit fluorometer, Agilent TapeStation 2200, and RT-qPCR using the Kapa Biosystems library quantification kit according to manufacturer's protocols. Uniquely indexed libraries were pooled in equimolar ratios and sequenced on an Illumina NextSeq500 with single-end 75 bp reads by the Dana-Farber Cancer Institute Molecular Biology Core Facilities. The total read depths for the three control-treated replicates were 61.4, 63.9, and 56.1 M. The total read depths for the three si-NRDE2-treated replicates were 57.8, 58.9, and 46.4 M. Sequenced reads were aligned to the hg19 reference genome assembly; >90% of reads were mappable in all samples. Gene counts were quantified using STAR (v2.5.1b) and differential expression testing was performed by DESeq2 (v1.10.1) as part of the VIPER analysis pipeline (https://bitbucket.org/cfce/viper/ ).
3
Total RNA Isolation and Quality Assessment
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Total RNA was isolated from tissue samples by using miRVana RNA isolation kit (ThermoFisher). RIN values were measured using Eukaryote total RNA assay on the Agilent 2100. RIN > 9 was regarded as high quality and sufficient for sequencing.
4
Cardiomyocyte Total RNA and microRNA Isolation and Quantification
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Total RNA and microRNA-enriched RNA were isolated from cultured cardiomyocytes with mirVana RNA isolation kit (Thermo Fisher, AM1561) using the manufacturer’s instructions and a described protocol [26 (link)].
Total RNA (2 µg/sample) was used to generate cDNA using Super Script II reverse transcriptase kit (Invitrogen, Carlsbad, CA, USA). RT-PCR was performed using primers presented inSupplementary Table S1 . RT-PCR cDNA products were used as templates for quantitative PCR (qPCR) assays, which were run in duplicates on StepOnePlus Real-Time PCR System using SYBR green PCR master mix (Invitrogen). Real-time PCR cycling conditions consisted of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. RT-PCR products were separated on 1.5% agarose gels, while the bands were quantified by densitometry using the LI-COR Odyssey imaging system and expressed relatively to the housekeeping genes U6 and β-actin.
Total RNA (2 µg/sample) was used to generate cDNA using Super Script II reverse transcriptase kit (Invitrogen, Carlsbad, CA, USA). RT-PCR was performed using primers presented in
5
RNA Isolation from Tissue and Serum
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For the small RNA sequencing, total RNA was isolated from tissue samples by using miRVana RNA isolation kit (ThermoFisher Scientific, Waltham, MA) following the total RNA procedure. RNA was eluted using RNase-free water and stored at − 80 °C. RNA from serum was isolated using the Qiagen Plasma/Serum kit (Qiagen, Hilden, Germany) using 200 μL of serum.
For the miRNA and mRNA microarray experiments, total RNA was isolated with TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA).
Exosomes were isolated from serum using size exclusion chromatography following the protocol of Böing et al. [32 ]. RNA was subsequently isolated using the above mentioned Qiagen Plasma/Serum kit.
For the miRNA and mRNA microarray experiments, total RNA was isolated with TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA).
Exosomes were isolated from serum using size exclusion chromatography following the protocol of Böing et al. [32 ]. RNA was subsequently isolated using the above mentioned Qiagen Plasma/Serum kit.
6
Transcriptome Profiling of Tumor and Normal Tissues
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Total RNA was isolated from 20 samples (collected from Henan Provincial People’s Hospital), including ten tumor tissues and ten adjacent normal tissues, using an mirVana™ RNA isolation kit (AM1561, Thermo Fisher, USA). RNA clean-up was performed using RNasey Mini Kit (Qiagen p/n 74,104). Quantification and quality control were achieved by NanoDrop ND-2100 (ThermoFisher) and Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, USA). cDNA synthesis and biotin labeling of cRNA were performed using One-Cycle Target Labeling and Control Reagents (Affymetrix, Santa Clara, USA) and the GeneChip™ Hybridization, Wash and Stain Kit (Affymetrix) following the manufacturer’s protocol. The purified and fragmented labeled cRNAs were hybridized onto the Affymetrix PrimeView™ Human Gene Expression microarray. After washing and staining, the arrays were scanned and analyzed using Affymetrix Scanner 3000 (Affymetrix). GeneChip Command Console Software (version 4.0, Affymetrix) and Genespring software (version 14.9, Agilent Technologies) were used to analyze the transcriptome data. Differentially expressed genes (DEGs) from TCGA were analyzed using DESeq2 package within R language (version 3.4.3).
7
RNA Isolation from Fibrin and Plasma
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A mirVana RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to isolate total RNA from fibrin. A mirVana PARIS Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to isolate RNA from 200 μL of plasma according to the manufacturer’s instructions. Briefly, recommended volume of miRNA homogenate additive (mirVana,) or 2X Denaturing Solution (PARIS) was added to each sample and mixed by vortexing. After adding 2X Denaturing Solution, plasma samples were spiked with synthetic cel-39 and incubated on ice. An equal volume of acid-phenol: and chloroform (Ambion) was added to each aliquot. The upper aqueous phase solutions were separated and mixed with 1.25 volumes of 100% molecular-grade ethanol. The solution was passed through a mirVana or PARIS column according to the manufacturer’s instructions. Finally, 100 µL of preheated 95 °C elution solution was used to elute total RNA. The concentration of total RNA isolated from fibrin was quantified using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) [32 (link),33 (link),34 (link)].
8
CRISPR-Cas9 sgRNA Synthesis in Zebrafish
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Crisprscan36 (link) was used to identify a target site in exon 3 of exosc9 in zebrafish. Single guide RNA (sgRNA) was produced as described elsewhere.37 (link) An oligonucleotide with a T7 promoter, exosc9 target sequence, and a complementary sequence (3′-taatacgactcactataGGGGGGCGTGAATCTTTTGGgttttagagctagaa-5′) was annealed to a bottom strand “ultramer” oligo (3′-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGG ACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-5′) in a thermocycler, and extension of the oligonucleotides was catalyzed by DNA polymerase (MyTaq) to form the template oligonucleotide for sgRNA synthesis.38 (link) The sgRNA template oligonucleotide was purified with a Qiagen PCR Purification Kit. sgRNA was synthesized from the sgRNA oligonucleotide template with the MEGAshortscript T7 Kit (Ambion), purified with the mirVana RNA Isolation Kit (ThermoFisher Scientific), and stored at −80°C until required for injection.
9
RNA-seq Analysis of Sorted and Bulk Cell Cultures
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For gene expression analysis, total RNA was collected using the mirVana RNA isolation kit (ThermoFisher Scientific) or RNeasy Mini Kit (Qiagen) from sorted (>20,000 cells) and bulk cultures (>1,000,000 cells). Illumina libraries were constructed using the TruSeq Stranded Total RNA with Ribo-Zero Globin (Illumina). Finally, libraries were quantified using Fragment Analyzer (Advanced Analytical). RNA-seq libraries were sequenced with HiSeq 4000 (Illumina) using a 2x76bp read length and alignment was performed using STAR Aligner92 (link) against the GRCh38 reference genome. Gene counts were obtained using featureCounts93 (link) and FPKM per gene were calculated using Cufflinks94 (link). Values were normalized using quantile normalization.
10
Extracting High-Purity Total RNA from Mice Soleus Muscle
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Total RNA was extracted from mice soleus muscle using miRVana RNA Isolation kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Total RNA concentration was measured using the NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Purity was determined by the 260/280 nm ratio, and the value of 2.0 indicated a high purity of our samples.
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