Total RNA extraction from tissues and cells was routinely performed by utilizing the TRIzol (Invitrogen, Carlsbad, CA, USA) method [30 (link)]. After the purity test, miRNA, LncRNA, and mRNA underwent reverse transcription into cDNA using the mirPremier® microRNA Isolation Kit (Sigma, St. Louis, MO, USA) and the PrimeScript RT kit (Invitrogen, Shanghai, China), respectively. Then, we adopted the SYBR®Premix-Ex-Taq™ (Takara, TX, USA) and the ABI7300 system for qRT-PCR. GAPDH served as a housekeeping gene to verify the mRNA levels of LncRNA MIR17HG, SNCA, IL-1β, interleukin-6 (IL-6), TH, and tumor necrosis factor (TNF-α). In contrast, U6 served as that of miR-153-3p. The primer sequences for each molecule are as follows. MIR17HG: F: 5’-TGTGCAGATTGAGCTCTCCT-3’, R: 5’-TCCTGACAAAATGCAGCCTG-3’; miR-153-3p: F: 5’-AATCGGCGTTGCATAGTCACAAA-3’; R: 5’-CAGTGCAGGGTCCGAGGT-3’; SNCA: F: 5’-GACTGGGCACATTGGAACTG-3’; R:5’-TGCCTGTGGATCCTGACAAT-3’; GAPDH: F: 5’-CTCCTCCTGTTCGACAGTCAGC-3’; R: 5’-CCCAATACGACCAAATCCGTT-3’. IL-1β: F: 5’-TCATCTTTTGGGGTCCGTCA-3’; R: 5’-GGCTCATCTGGGATCCTCTC-3’; IL-6: F: 5’-TTTCACCAGGACCGTCTCTCCT-3’; R:5’-AGACAGCCACTCACCTCTTC-3’; TNF-α: F: 5’-ATCCCAGGTTTCGAAGTGGT-3’; R: 5’-TCTGGGCAGGTCTACTTTGG-3’; TH: F: 5’-GCGTGGACAGCTTCTCAATT-3’; R: 5’-TGTTCCAGTGCACCCAGTAT-3’. U6: F: 5’-CTCGCTTCGGCAGCACATATACTA-3’; R:5’-ACGAATTTGCGTGTCATCCTTGC-3’.
Mirpremier microrna isolation kit
Manufactured by Merck Group
Sourced in United States, Germany
The MirPremier microRNA Isolation Kit is a laboratory product designed to extract and purify microRNA (miRNA) from a variety of sample types, including cells, tissues, and body fluids. The kit utilizes a silica-based membrane technology to selectively capture and isolate miRNA molecules, allowing for their subsequent analysis and study.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Mir x mirna first strand synthesis kit, by Takara Bio (3 mentions)
Miscript kit, by Qiagen (3 mentions)
Taqman universal master mix 2, by Thermo Fisher Scientific (3 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Mmessage mmachine t7 transcription kit, by Merck Group (2 mentions)
Mmessage mmachine t7 transcription kit, by Qiagen (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Sybr green real time master mix, by Thermo Fisher Scientific (2 mentions)
Cdna matermix, by Takara Bio (2 mentions)
Lightcycler faststart dna masterplus sybr green 1 kit, by Roche (2 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (2 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Multidoc it, by Analytik Jena (2 mentions)
Truseq small rna sample preparation, by Illumina (2 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
47 protocols using mirpremier microrna isolation kit
1
Multiplex qRT-PCR Analysis of RNA Species
2
Rapid RNA Extraction from Plant Leaves
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Combined total plant RNA and small RNA extractions were performed using a single leaf that was snap-frozen in liquid nitrogen, homogenized, divided into two approximately 50 mg aliquots and processed with a Thermo Scientific GeneJET Plant RNA Purification Mini Kit (Thermo Scientific, Waltham, MA, USA) and mirPremier microRNA Isolation Kit (Sigma-Aldrich, St. Louis, MI, USA), respectively, following manufacturers’ recommendations. The quantification and quality control of the RNA extracts were performed using a Nanodrop 1000 UV-Vis spectrophotometer and Qubit HS RNA and IQ assays (Invitrogen, Carlsbad, CA, USA).
3
PBMC miRNA Extraction and cDNA Synthesis
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Total RNA, including miRNAs, was extracted from PBMCs using the mirPremier™ microRNA Isolation Kit (Sigma, Germany) following the manufacturer’s instructions. The RNA concentration and purity were quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized by using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystem, USA) containing RNA-specific RT primer according to the manufacturer’s instruction.
4
RNA Extraction and Expression Analysis
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For RNA extraction, subcutaneous tumors from mice and patient tissues were snap-frozen and homogenized. RNA was isolated from A549 and H1229 cells with indicated transfection and tissue homogenates with TRIzol Reagent from Sigma (St. Louis, MO, USA) and quantified with Qubit 4 (ThermoFisher, Waltham, MA, USA), which was reversely transcribed into cDNA. The mirPremier microRNA Isolation Kit from Sigma (St. Louis, MO, USA) was used to extract miRNAs, and miRNAs were reversely transcribed using the miScript kit (QIAGEN, Germantown, MD, USA). The expression of circ_0015278, miR-1278, and SOSC6 was examined by quantitative real-time PCR. Circ_0015278 and SOCS were normalized to GAPDH, and miR-1278 was normalized to U6 snRNA. Primers used are listed in Table 2 .
5
CRISPR-Mediated lncRNA Knockout in Mice
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LncRNA knockout models were produced in the Transgenic Unit of the Institute of Molecular Genetics ASCR, Czech Centre for Phenogenomics using Cas9-mediated deletion of lncRNA promoters (Supplementary Figs. S7 and S8 ).15 (link),16 All animal experiments were approved by the Institutional Animal Use and Care Committees (project number 58-2015) and were carried out in accordance with the law.
Sequences of guide RNAs are listed in the Table S5. To produce guide RNAs, synthetic 128 nt guide RNA templates including T7 promoter, 18 nt sgRNA and tracrRNA sequences were amplified using T7 and TracrRNA primers (Supplementary Table S5 ). Guide RNAs were produced in vitro using the Ambion mMESSAGE mMACHINE T7 Transcription Kit, and purified using the mirPremier microRNA Isolation Kit (Sigma). The Cas9 mRNA was synthesized from pSp Cas9-puro plasmid using Ambion mMESSAGE mMACHINE T7 Transcription Kit, and purified using the Qiagen RNasy mini kit. A sample for microinjection was prepared by mixing two guide RNAs in ultra-pure water at a concentration of 25 ng/µl for each one together with Cas9 RNA (100 ng/µl) . Five picoliters of the microinjection mixture were injected into male pronuclei of C57Bl/6 zygotes and transferred into pseudopregnant recipient mice. PCR genotyping was performed on tail biopsies from 4 weeks-old animals. Primers are listed in Supplementary Table S5 .
Sequences of guide RNAs are listed in the Table S5. To produce guide RNAs, synthetic 128 nt guide RNA templates including T7 promoter, 18 nt sgRNA and tracrRNA sequences were amplified using T7 and TracrRNA primers (
6
Quantitative Analysis of miR-29 Family in Dermal Samples
7
Generation of Sirena1 Knockout Mouse
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The Sirena1 deletion mutant model was produced in the Czech Centre for Phenogenomics at the Institute of Molecular Genetics ASCR using Cas9-mediated deletion of the Sirena1 promoter (13 (link)). Sequences of guide RNAs are listed in Supplementary Table S1 . To produce guide RNAs, synthetic 128 nt guide RNA templates including T7 promoter, 18nt sgRNA and tracrRNA sequences were amplified using T7 and tracrRNA primers. Guide RNAs were produced in vitro using the Ambion mMESSAGE mMACHINE T7 Transcription Kit, and purified using the mirPremier™ microRNA Isolation Kit (Sigma). The Cas9 mRNA was synthesized from pSpCas9-puro plasmid using the Ambion mMESSAGE mMACHINE T7 Transcription Kit, and purified using the RNeasy Mini kit (Qiagen). A sample for microinjection was prepared by mixing two guide RNAs in water (25 ng/μl for each) together with Cas9 mRNA (100 ng/μl). Five picoliters of the mixture were microinjected into male pronuclei of C57Bl/6J zygotes and transferred into pseudo-pregnant recipient mice. PCR genotyping was performed with tail biopsies from four-week-old animals (primers are listed in Supplementary Table S1 ). We obtained seven positive founders, of which one transmitted the mutant allele to F1. After two generations of breeding with C57Bl/6NCrl animals, the heterozygotes were used for breeding Sirena1−/− animals for phenotype analysis.
8
Profiling miRNA expression via qRT-PCR
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All primers were provided from Bioneer, South Korea. MirPremier microRNA isolation kit was sourced from Sigma-Aldrich, USA. Mir-X miRNA First-Strand Synthesis kit and cDNA matermix were purchased from Takara bio inc, USA. SYBR® Green Real-Time Master Mix was from Invitrogen, UK.
9
Profiling circTMCO3 and miR-515-5p Regulation
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RNA was isolated using Total RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) and reversely transcribed into cDNA. The mirPremier microRNA isolation kit (Sigma) was used to isolate miRNAs, which were reversely transcribed using the miScript kit (QIAGEN, Germantown, MD, USA). Quantitative PCR was applied to detect circTMCO3, TMCO3, miR-515-5p, ITGA8, TNF-α, iNOS, IL-10, Arg-1 using SYBR Green (Beyotime). GAPDH and U6 snRNA were used as normalization controls. The 2−∆∆Ct method was used to calculate their relative expression. Primers were listed in Supplementary Table 1 .
10
Purification and Isolation of miRNA
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Total RNA was extracted 72 h after transfection from E10 cells transfected with miRNA mimics or scrambled control, and from C12, HTC116, and HT29 cells, using the RNeasy Mini Kit as described by the manufacturer (Qiagen, Hilden, Germany). The RNA samples were treated with the TURBO DNA-free kit (Life Technologies Ltd, Paisley, UK) to remove contaminating genomic DNA.
RNA-fractions enriched with respect to miRNAs were isolated according to the manufacturer's protocol from transfected E10 cells using the mirPremier microRNA isolation kit (Sigma-Aldrich, St. Louis, MO, USA).
Concentrations of solutions of purified RNA were assayed using the Nanodrop ND-1000 spectrophotometer. RNA fractions exhibiting a ratio of OD260/OD280 and OD260/OD230 of at least 1.8 and 2.0, respectively, were used for further analysis.
RNA-fractions enriched with respect to miRNAs were isolated according to the manufacturer's protocol from transfected E10 cells using the mirPremier microRNA isolation kit (Sigma-Aldrich, St. Louis, MO, USA).
Concentrations of solutions of purified RNA were assayed using the Nanodrop ND-1000 spectrophotometer. RNA fractions exhibiting a ratio of OD260/OD280 and OD260/OD230 of at least 1.8 and 2.0, respectively, were used for further analysis.
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