qRT-PCR was used to detect the RNA expression of METTL14 and miR-1306-5p. All the RNA was collected from blood, cell, or tissue samples using the miRNeasy extraction kit (QIAGEN). For quantitative analysis, the cDNA was reversed by miRNA Reverse Transcription Kit (MR101-01/02, Vazyme) and detected by all-in-one miRNA RT-qPCR Detection Kit (Q711-02, Vazyme) with U6 as the internal control. As for METTL14 mRNA detection, TRIzol (BS259A, Biosharp) was used for RNA isolation and PrimeScript RT Reagent kit (R223-01, Vazyme) was used to reverse RNA into cDNA. SYBR Green Real-Time PCR Master Mix (Q711-02, Vazyme) was used for RT-PCR assay with β-actin as the control. The primers for miR-1306-5p, U6, METTL14, and β-actin were listed as below: METTL14, sense, 5′- GAGTGTGTTTACGAAAATGGGGT-3′; antisense, 5′- CCGTCTGTGCTACGCTTCA-3′; β-actin: sense, 5′-AGCGAGCATCCCCCAAAGTT-3′, antisense: 5′-GGGCACGAAGGCTCATCATT-3′; U6: sense, 5′-CTCGCTTCGGCAGCACA-3′, antisense: 5′-AACGCTTCACGAATTTGCGT-3′; miR-1306-5p reverse primer: 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTGGACGTT-3′; miR-1306-5p sense: 5′-AATACCACCTCCCCTGCA-3′. 2−ΔΔCt method was used for analysis of relative expression.
Mirneasy extraction kit
Manufactured by Qiagen
Sourced in United States, Germany
The MiRNeasy extraction kit is a product by Qiagen designed for the isolation and purification of total RNA, including small RNA species such as microRNA (miRNA), from a variety of sample types. The kit utilizes a silica-membrane-based technology to efficiently capture and purify the RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (18 mentions)
Miscript 2 rt kit, by Qiagen (10 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (8 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
Rt2 first strand kit, by Qiagen (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Quantitect reverse transcription kit, by Qiagen (3 mentions)
Cel mir 39, by Qiagen (3 mentions)
Hiseq 2000, by Illumina (3 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Random hexamers primers, by Roche (2 mentions)
Dnase, by Promega (2 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
First strand cdna synthesis kit, by Roche (2 mentions)
Reverse transcription kit, by GenePharma (2 mentions)
Rotor gene q system, by Qiagen (2 mentions)
74 protocols using mirneasy extraction kit
1
Quantifying METTL14 and miR-1306-5p in Biological Samples
2
RNA Extraction and Integrity Analysis
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Total RNA was extracted using the miRNeasy extraction kit (Qiagen, Hilden, Germany) and DNase treated using DNA-free™ (Thermo Fisher Scientific) as described13 (link). RNA was quantified using the nano-drop spectrophotometer and integrity analyses was performed using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara CA). RNA with an RIN > 7 was used for further analysis. RNA was stored at −80 °C until further use.
3
miRNA and lncRNA Extraction and Analysis
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MiRNeasy extraction kit (Qiagen, Hilden, Germany) was used to get the total RNA (including microRNAs and lncRNAs) extracted from the serum samples after adding the QIAzollysis reagent following the manufacturer’s protocol. The extracted RNA was determined using the NanoDrop® (ND)-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA).
Total RNA was reverse-transcribed using the RT2 first strand Kit (Qiagen, Maryland, MY, USA) in a whole volume of 20 μL/reaction for the long noncoding RNA analysis, while the miScript II RT kit (Qiagen) was used for the miRNA analysis in a 20-μL RT reaction according to the manufacturer’s instructions.
Total RNA was reverse-transcribed using the RT2 first strand Kit (Qiagen, Maryland, MY, USA) in a whole volume of 20 μL/reaction for the long noncoding RNA analysis, while the miScript II RT kit (Qiagen) was used for the miRNA analysis in a 20-μL RT reaction according to the manufacturer’s instructions.
4
Total RNA Extraction and Quantification
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We performed total RNA extraction from the whole body or specific tissues if indicated, using the miRNeasy extraction kit (QIAGEN). A 500-ng sample from each RNA extraction was treated with DNase (Promega) and reverse transcribed with first Strand cDNA Synthesis Kit (Roche) and random hexamers primers (Roche). RNA quantity and quality was estimated by spectrophotometric absorption at 260 nm using a Nanodrop Spectrophotometer ND-1000 (NanoDrop Technologies).
5
Quantification of miR-125a-5p and TAZ in Colorectal Cancer
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Total RNA was extracted from LoVo and SW480 cells using Trizol reagent (Invitrogen, USA) and miRNeasy extraction kit (Qiagen; Germantown, MD) according to the manufacturer’s instructions. Single-stranded cDNA was synthesized with QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA). Quantitative RT-PCR (qRT-PCR) was performed in 35 cycles using SYBR Green PCR Master mix (Takara Bio, Inc., Shiga, Japan). Each cycle consisted of 30 seconds at 94°C, 30 seconds at 60°C and 30 seconds at 72°C. TaqMan Universal PCR Master mix (all from Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for the quantification of the miR-125a-5p. RT-qPCR was performed with the Bio-Rad CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA). GAPDH and U6 were used as the internal control. The primer sequences used were as follows: miR-125a-5p forward, 5ʹ-GGTCATTCCCTGAGACCCTTTAAC-3ʹ; reverse, 5ʹ-GTGCAGGGTCCGAGGT-3ʹ. TAZ forward, 5ʹ-ACCCACCCACGATGACCCCA-3ʹ; revere, 5ʹ-GCACCCTAACCCCAGGCCAC-3ʹ; GAPDH, forward, 5ʹ-GGAGCCAAAAGGGTCATCAT-3ʹ; revere, 5ʹ-GTGATGGCATGGACTGTGGT-3ʹ. U6, forward, 5ʹ-CTCGCTTCGGCAGCACA-3ʹ; 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ. The qRT-PCR experiments were performed in 3 independent experiments, each of which used 3 independent samples. 2−∆∆Ct method was used to calculate the relative expression of each gene.
6
Serum microRNA Extraction Protocol
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RNA with microRNAs was extracted from 200 μl serum using the miRNeasy extraction kit (Qiagen, Valencia, CA, USA), 1,000 μl QIAzol lysis reagent, and 5 min at room temperature. Chloroform (200 μl) was applied, vortexed for 15 s, and then incubated at room temperature for 2–3 min. After that, centrifugation at 12,000×g for 15 min at 4°C was performed. The upper watery phase was isolated and 1.5 times its volume was applied to ethanol (100%). Then 700 μl of this mixture was centrifuged (8,000×g) at RT for 15 s in an RNeasy Mini spin column in a collection tube (2 ml). After the mixture had passed through the column full, 700 μl of RWT buffer was applied to each column and centrifuged again (at 8,000×g; at RT) for 15 s. The column was loaded with 500 μl of buffer RPE (centrifuged; 8,000×g; RT; 15 s). The column was moved to a new 1.5-ml collection tube and 50 μl RNase-free water was applied to the column before centrifuging for 1 min at 8,000×g to elute RNA.
7
Quantification of Plasma microRNAs
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For qRT-PCR, a synthetic Caenorhabditis elegans microRNA, cel-miR-39 (Qiagen, Valencia, CA, USA), was added to plasma samples as a control prior to RNA extraction. Total RNA was extracted from 200 μL individual plasma samples with a miRNeasy extraction kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. RNA (6 μl) was reverse transcribed into cDNA (in a final volume of 10 μl) using a reverse transcription kit (GenePharma, Shanghai, China). Subsequently, quantitative real-time PCR was performed on an iQ™5 Real-Time PCR Detection System (Bio-Rid, USA) using SYBR Green. The primers used for qRT-PCR are listed in Table 2 . Relative microRNA levels were determined by the ΔΔCT method.
8
Quantitative RT-PCR Analysis of gene expression
9
Serum RNA Extraction and Quantification
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By utilizing a miRNeasy extraction kit (Qiagen, Valencia, CA, USA), total RNA was isolated from the serum. RNA's quantity and quality were assessed using the NanoDrop™ 2000 (Thermo Scientific, USA).
10
qRT-PCR Analysis of Plasma miRNAs
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For qRT-PCR, a synthetic Caenorhabditis elegans microRNA, cel-miR-39 (Qiagen, Valencia, CA, USA), was added to plasma samples as a control prior to RNA extraction. Total RNA was extracted from 200 μL individual plasma samples with a miRNeasy extraction kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. RNA (6 μl) was reverse transcribed into cDNA (in a final volume of 10 μl) using a reverse transcription kit (GenePharma, Shanghai, China). Subsequently, quantitative real-time PCR was performed on an iQ™5 Real-Time PCR Detection System (Bio-Rid, USA) using SYBR Green. The primers used for qRT-PCR are listed in Table 1 . Relative microRNA levels were determined by the 2−ΔCT method.
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