Expression of miR-320e in the validation cohort was performed utilising the TaqMan reverse transcription–PCR (qRT–PCR) method with the predesigned TaqMan miRNA expression assays (Applied Biosystems Inc., Foster City, CA, USA) using a StepOnePlus Real-Time PCR System (Applied Biosystems). All the experiments were done in duplicate. Results were expressed as 2−ΔCt, and the results were normalised to miR-16. To keep consistent measurements throughout all plates, two independent RNA cell line samples were loaded as internal controls along with each PCR run, and the results from each plate were normalised according to data obtained from these internal controls.
Taqman mirna expression assays
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan miRNA expression assays are a set of laboratory tools used for the quantitative analysis of microRNA (miRNA) expression levels. These assays employ real-time PCR technology to provide accurate and sensitive measurement of miRNA expression in biological samples.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (4 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Mirna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Abi 7900ht, by Thermo Fisher Scientific (1 mentions)
Sybr green realtime pcr master mix, by Toyobo (1 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
6 protocols using taqman mirna expression assays
1
Quantification of miR-320e Expression
2
RNA Extraction and miRNA cDNA Synthesis
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After collection, cells were washed with PBS and lysed with Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's specifications for RNA extraction. Final, purified RNA was dissolved in sterile distilled water. cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA), according to the manufacturer's protocol and using 400 ng of RNA per reaction. To obtain miRNA-specific cDNA, random primers were replaced with the corresponding TaqMan miRNA-Expression Assays (Applied Biosystems, Life Technologies, Carlsbad, CA).
3
mRNA and miRNA Expression Analysis
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qRT-PCR was performed on an ABI 7900HT instrument (Applied Biosystems, USA) with SYBR Green Real-time PCR Master Mix (Toyobo, Japan). For mRNA detection, glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used for internal normalization. The primers used for mRNA detection are listed in Supplementary Table S1. For miRNA detection, reverse transcription and miRNA detection were carried out using the miRNA Reverse Transcription kit and TaqMan miRNA Expression Assays (Applied Biosystems, USA). Small nuclear RNA U6 was used for internal normalization.
4
Quantifying miRNA Expression in Cells
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Total RNA from cells or homogenized tissues was extracted using Trizol (Invitrogen) following manufacturer’s instructions. Expression of miRNAs was determined with TaqMan miRNA expression assays (Applied Biosystems) according to the manufacturer's instructions. Assays were performed on a minimum of three biological replicates and normalized to sno135.
5
Quantifying miRNA Expression in Cells
6
Quantitative RT-PCR for Gene and miRNA Expression
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cDNA was synthesized from 1 μg of Trizol-extracted (Life Technologies, Carlsbad, CA) total RNA using QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Quantitative cDNA amplification was carried out in triplicate for each sample in a final reaction volume of 10 μL, using 4.5 μL of cDNA (1/10 diluted), 5 μL Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA), and 0.5 μL of 10-μM specific oligonucleotides for the gene of interest (Sigma, St. Louis, MO), in an ABI Prism 7300 sequence detector system with standard settings. Relative expression was calculated as RQ = 2−ΔΔCt. The oligonucleotides are indicated in S2 Table . As an internal control, gene expression was normalized to the mouse Actb, Gapdh, or 18S genes. miR-29 detection was performed using Taqman miRNA expression assays (Applied Biosystems, Foster City, CA) for the double KO mice experiments or Taqman Advance miRNA expression assays (Applied Biosystems, Foster City, CA) for the single KO mice experiments. As an internal control, miR-29 expression was normalized against snoRNA202 (Taqman miRNA Assays) or 18S (Taqman Advance miRNA assays). All protocols were carried out according to the manufacturer’s instructions.
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