For the gene expression analysis, total RNA was extracted with the MiniBEST Universal RNA Extraction Kit (TaKaRa, Tokyo, Japan) or the miRNA Mini Kit (QIAGEN, Dusseldorf, Germany) in strict accordance with the instructions of the manufacturers. PrimeScript™ RT reagent (TaKaRa) was used for mRNA reverse transcription, and a miRNA First Strand cDNA Synthesis Tailing Reaction Kit (B532451, Sangon Biotech, Shanghai, China) was utilized for miRNA reverse transcription. After that, the TB Green™ Premix Ex Taq™ II kit (TaKaRa) was utilized to perform qRT‐PCR on a Real‐Time PCR Detection System (Bio‐Rad). U6 was utilized to normalize the miRNA expression levels. For the analysis of miRNA expression in EVs, samples were spiked with cel‐miR‐39 (miRB0000010‐3‐1, RIOBO) to control inter‐sample variability as described in previous studies.18 For mRNA analysis, β‐actin was employed to normalize the levels of genes of interest. The sequences of all primers used in the present study are shown in Table S1 .
Mirna mini kit
Manufactured by Qiagen
Sourced in Germany, United States
The MiRNA Mini Kit is a laboratory equipment product designed for the purification of microRNA (miRNA) from various sample types. It is a tool used in scientific research and analysis, providing a standardized method for extracting and isolating miRNA molecules from cells, tissues, or other biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt master mix, by Takara Bio (3 mentions)
Nuclear and cytoplasmic extraction reagents kit, by Beyotime (2 mentions)
Mir xtm mirna first strand synthesis kit, by Takara Bio (2 mentions)
Mrq 3 primer, by Takara Bio (2 mentions)
Cfx96 real time qpcr instrument, by Bio-Rad (2 mentions)
Nanodrop nd 1000 spectrophotometer, by Agilent Technologies (2 mentions)
Lightcycler 480 2, by Roche (2 mentions)
Sybr premix ex taqtm 2, by Takara Bio (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Minibest universal rna extraction kit, by Takara Bio (1 mentions)
Primescript rt reagent, by Takara Bio (1 mentions)
Real time pcr detection system, by Bio-Rad (1 mentions)
Tb green premix ex taq 2 kit, by Takara Bio (1 mentions)
Mirna first strand cdna synthesis tailing reaction kit, by Sangon (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Iscript advanced cdna synthesis kit, by Bio-Rad (1 mentions)
Truseq small rna kit, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq 2000 sequencer, by Illumina (1 mentions)
Abi viiatm7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
15 protocols using mirna mini kit
1
Gene Expression Analysis Protocol
2
Bone Marrow Macrophage Gene Expression
3
Production and Purification of 5'pppRNA
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The sequence of 5′pppVSV hairpin RNA was derived from the 5′ and 3′ UTRs of the VSV genome as previously described (Schlee 2009 Immunity) and in vitro transcribed RNA was prepared as previously described (Goulet 2013 PLoS Pathogens). CVB3 ivtRNAs were produced by T7-driven in vitro transcription using PCR products amplified the CVB3 infectious clone p53CB3/T7 [26] (link). 5′pppRNAs were purified using Qiagen miRNA Mini Kit (Qiagen) or by precipitation. Integrity and concentration of purified RNA ligands were controlled by agarose gel electrophoresis prior to each transfection experiment.
4
Small RNA Sequencing of Salt-Stressed Plants
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Leaf and root tissues from 6 control and salt-treated seedlings were separately pooled, and small RNAs were isolated using the miRNA Mini Kit (Qiagen). The quality and quantity of the RNA samples were tested using an Agilent 2100 Bioanalyzer (Agilent). The small RNAs were ligated with 3′ and 5′ adaptors, and RT-PCR was performed using the TruSeq™ Small RNA kit (Illumina) according to the manufacturer's instructions. The expected final PCR product was isolated from the gel, purified and sequenced using Hiseq2000 sequencer (Illumina). sRNA sequencing and basic bioinformatics analyses were conducted at Macrogen, Korea and LC Sciences, Texas, USA, respectively.
5
Comprehensive RNA Extraction and qPCR Analysis
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Total cellular RNA was extracted using the miRNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Nuclear RNA and cytoplasmic RNA were extracted following the instructional manuals of the nuclear and cytoplasmic extraction reagents kit (Beyotime, Beijing, China). cDNA was synthesized from miRNA and mRNA using the Mir-XTM miRNA First-Strand Synthesis Kit (Takara Bio Inc., Shiga, Japan) and a PrimeScriptTM RT Master Mix (Takara Bio Inc., Shiga, Japan), respectively. PCR reaction was conducted in 20 μl total volume containing a final concentration of 0.5 mM of each primer, 6.4 μl ddH2O, 10 μl of 2x SYBR Premix Ex TaqTM II (Takara Bio Inc., Shiga, Japan) and 2 μl of cDNA sample (1:20 diluted) corresponding to 1 μg of total RNA. RT-qPCR reaction was performed on an ABI ViiATM 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, United States). The amplification products were detected by agarose gel electrophoresis and sequencing. Transcript levels of lncRNA and mRNA were normalized to that of the GAPDH housekeeping gene, and those of miRNA were normalized to that of the small U6 RNA. The RT-qPCR primer sequences are listed in Supplementary Table 1 . Expression levels were calculated using the 2–ΔΔCt method (Livak and Schmittgen, 2001 (link)). Each experiment was performed in triplicate.
6
RNA Extraction and Quality Assessment
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The iPS and iPS-RPE cells were lysed by running the samples through a Qi Shredder column (Qiagen, Valencia, CA, USA). Total RNA was extracted using the miRNA mini kit (Qiagen, Valencia, CA, USA),35 followed by checking with a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA) for the concentration. RNA samples without organic solvent carryover, showing OD 260/230 above 1.7 were recommended to proceed for RNA quality determination. The quality of the RNA was determined with Eukaryote Total RNA Pico kit (Agilent Technologies, Santa Clara, CA) performed on a 2100 Bioanalyzer and software (Agilent Technologies) that detects 28S and 18S ribosomal RNA ratio and total RNA Integrity Number (RIN). The RIN software algorithm allows the classification of total RNA, based on a numbering system from 1 to 10, with 1 being the most degraded and 10 being the most intact. Only samples with 28S and 18S ribosomal RNA ratio higher than 1.8 and RIN higher than 8 were used in this study.
7
Chondrocyte RNA Extraction and RT-qPCR
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RNA extraction and reverse transcription were performed as described in our previous study31 (link). Briefly, total RNA was extracted from the isolated chondrocytes using the miRNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Nuclear and cytoplasmic RNA were extracted following the instructions of the Nuclear and Cytoplasmic Extraction Reagents Kit (Beyotime Biotechnology, Beijing, China). RT‒qPCR was performed using an ABI ViiA™ 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The specific primers used for these analyses are listed in Supplementary Table 1 . Gene expression was calculated using the 2-ΔΔCt method32 (link).
8
High-Throughput TCR β Sequencing
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HTS for TCR β chain was performed on PBMCs, enriched CD8+ T cells, sorted Tax11-19–specific CD8+ T cells and CSF cells. The TCR β chain was amplified from 2 × 106 cells of PBMCs and enriched CD8+ T cells. The sorted Tax11-19–specific CD8+ T cells and CSF cells were used at 4 × 104 to 5 × 105 cells and 1 × 104 to 2.5 × 105 cells, respectively, for the amplification of TCR β chain. Total RNA was extracted from PBMCs and enriched CD8+ T cells using a miRNA Mini Kit (Qiagen), and RNA from sorted Tax11-19–specific CD8+ T cells and CSF cells was extracted using a RNeasy Micro kit (Qiagen) following the manufacturer’s instructions. The rearranged TCR β repertoires were amplified using previously described methods (14 (link)). Briefly, cDNA synthesis was performed using the anchored switch 5′ rapid amplification of cDNA ends (5′ RACE) PCR-based primer combined with a unique molecular barcode that consists of 10 random nucleotide sequences. Nested PCR was performed using a pair of primers specific for the TCR constant region and 5′ RACE region, which contains a sample index and Illumina adapter sequences. Final PCR product was purified, and the quality of TCR library was analyzed using a Bioanalyzer (Agilent Technologies).
9
Analyzing miRNA and mRNA Expression
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Cartilage and cell-seeded scaffolds were ground in liquid nitrogen prior to RNA isolation. Total RNA from cells, cartilage samples, and cell-seeded scaffolds was extracted using a miRNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions.
Next, cDNA was synthesized from miRNA and mRNA using a Mir-X™ miRNA First-Strand Synthesis Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) and a PrimeScript™ RT Master Mix (Takara, Shiga, Japan), respectively. qRT-PCR of target genes was performed using SYBR® Premix Ex Taq™ II (Takara) and a CFX96 real-time qPCR instrument (Bio-Rad, Hercules, CA, USA), according to the manufacturer's instructions. Transcript levels were normalized to that of the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for mRNA, the small U6 RNA for miRNA, or miR-39 for exosomal miRNA. The specific primers used for these analyses are shown inSupplementary Material . The mRQ 3′ Primer (Clontech) was used as the reverse primer for miRNA-193b-3p, and the miR-39 primer was supplied in the miRNeasy Serum/Plasma Kit. Gene expression was calculated using the 2-ΔΔCt method, and each experiment was performed in triplicate.
Next, cDNA was synthesized from miRNA and mRNA using a Mir-X™ miRNA First-Strand Synthesis Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) and a PrimeScript™ RT Master Mix (Takara, Shiga, Japan), respectively. qRT-PCR of target genes was performed using SYBR® Premix Ex Taq™ II (Takara) and a CFX96 real-time qPCR instrument (Bio-Rad, Hercules, CA, USA), according to the manufacturer's instructions. Transcript levels were normalized to that of the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for mRNA, the small U6 RNA for miRNA, or miR-39 for exosomal miRNA. The specific primers used for these analyses are shown in
10
Profiling Glioma miRNA Signatures
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Three tissue samples each from glioma patients and healthy controls were sent to Shanghai Kangcheng Biotechnology Co. Ltd., using the miR-CURY LAN Array system (Exiqon, Vedbaek, Denmark) for miRNA microarray analysis. Briefly, total RNA was extracted from the tissues using Trizol reagent, and purified with a miRNA Mini kit (Qiagen, #217004, Germany) according to the instructions. The purity and concentration of the RNA samples were analyzed using a spectrophotometer, and 1μg RNA per sample was labeled using a Hy3/Hy5 Power Labeling kit (Exiqon, Vedbaek, Denmark) according to the instructions. The labeled RNA was then hybridized with a miRCURY tm LNA Array (v.19.0, Exiqon) according to array manual. After hybridization, the sliced were obtained and washed using Wash buffer kit (Exiqon). The original signal intensity of the chip was tested using a GenePix 4000B chip scanner (MD). Using intensity (int) > 50 as the normalization factor, the differences in miRNA expression levels between the glioma and para-cancer tissues were analyzed by inter-chip standardization, intra-chip standardization, expression difference comparison, statistical significance test, and cluster analysis. Following normalization, the miRNA with significant differential espression between two groups were determined by Folding changes ( > 2) and P values ( < 0.05).
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