Total RNA was isolated from tissue using Trizol (Invitrogen 15596018). Reverse transcription was carried out using Biorad iScript cDNA synthesis kit (170-8891). Quantitative PCR reactions were performed on Applied Biosystems 7,000 detection system using Biorad iTaq Universal SYBR green supermix (172-5124). Normalization was based on the expression of beta-actin and relative gene expression or relative mRNA level was determined using the deltadelta cycle threshold method. Primers used for quantitative reverse transcription–PCR analysis are available upon request. For RNA-seq, total RNA was isolated from tissue using miRNAeasy (Qiagen 217004) and on column DNAseI digestion was performed (Qiagen 79254).
Mirnaeasy
Manufactured by Qiagen
Sourced in United States
The MiRNAeasy is a lab equipment product designed for the isolation and purification of microRNA (miRNA) from various sample types. It provides a reliable and efficient method to extract miRNA, which are small non-coding RNA molecules involved in gene expression regulation.
Automatically generated - may contain errors
Lab products found in correlation
Taqman universal mmixii, by Thermo Fisher Scientific (2 mentions)
Taqman validated assays, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
G2565ba scanner, by Agilent Technologies (1 mentions)
Mirnaeasy ffpe kit, by Qiagen (1 mentions)
Qiamp dna kit, by Qiagen (1 mentions)
Nucleospin mirna plasma, by Macherey-Nagel (1 mentions)
Mrnaeasy, by Qiagen (1 mentions)
Mirscript cdna synthesis kit, by Qiagen (1 mentions)
6 protocols using mirnaeasy
1
Quantitative Gene Expression Analysis
2
Quantitative Real-Time PCR of miR-34 Family
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Total RNA was purified by miRNAeasy (Qiagen, Germantown, MD, USA) and reverse-transcribed using TaqMan UNIVERSAL MMixII (Applied Biosystems, Waltham, MA, USA) for random priming or MicroRNA (miRNA)-specific assay reverse transcription. Semiquantitative PCR was performed with TaqMan-validated assays (Applied Biosystems): miR34a (000426), hsa-miR-34b (000427), miR34c (000428), hsa-miR-34c-3p (241009_mat). As reference for cDNA, we chose GAPDH (Hs99999905_m1) and U6 (#001973) for miRNA. All analyses were carried out in triplicate. Real-time data were collected using Microsoft Excel, and analyzed with the following formula: Expression level = 2−ΔΔCt method. All experiments were done as independent triplicates and analyzed using standard deviation (SD). The p-value was obtained with the Student’s t-test.
3
Microarray Analysis of miRNA Expression
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Total RNA was purified by miRNAeasy (Qiagen, Germantown, MD, USA) and retrotranscribed using the Agilent miRNA Labeling Reagent and Hybridization Kit small RNA Agilent—Human miRNA Microarray Kit (V3), and hybridized with the SurePrint Array HD G4470C Microarray Human miRNA Kit (V3) 8x15K containing 866 human and 89 human viral miRNAs (Release 12.0). After hybridization, the microarray was washed according to the manufacturer's protocol. The chips were scanned with an Agilent G2565BA scanner.
Processed logarithmic signal intensities were normalized by quantile normalization method. In each sample, the top‐expressed miRNA covering 90% of the whole miRNA expression was selected, and cluster analysis was performed by considering their union (n = 93). Cluster analysis was implemented with average linkage function. Microarray expression profile data, obtained from the same samples, were compared by computing Pearson's correlation.
Processed logarithmic signal intensities were normalized by quantile normalization method. In each sample, the top‐expressed miRNA covering 90% of the whole miRNA expression was selected, and cluster analysis was performed by considering their union (n = 93). Cluster analysis was implemented with average linkage function. Microarray expression profile data, obtained from the same samples, were compared by computing Pearson's correlation.
4
Extraction and Quantification of DNA and miRNA from Glioblastoma Cells and Tissues
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DNA from HBT-14 (U-87 MG) cells was extracted using the QIAmp DNA kit (Qiagen™) according to the manufacturer’s recommendation.
The miRNAs from HBT-14 (U-87 MG) cells were extracted using miRNAeasy (Qiagen™). The miRNAs from the 97 FFPE (formalin-fixed paraffin-embedded) surgically resected tumor specimens or the 8 non-tumoral brain tissues were extracted on 3 adjacent 15 μm cuts using the miRNAeasy-FFPE kit (Qiagen™, Hilden, Germany). For the tumoral specimens, morphological control was systematically carried out beforehand by a neuropathologist (ELZ) in order to guarantee that the percentage of tumor cells was greater than 70% and the absence of areas of necrosis or hemorrhage. When this was not the case, a macro-dissection of the samples was performed to determine these quality criteria. miRNAs were extracted from the seven plasma samples using NucleoSpin miRNA Plasma (Macherey-Nagel™, Düren, Luxembourg). For each sample, miRNA extraction was carried out according to the respective manufacturer’s instructions.
The integrity and quality of the purified DNA were assessed by 1% agarose gel electrophoresis, and the DNA/miRNA concentration was measured with the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Asnières-sur-Seine, France).
The miRNAs from HBT-14 (U-87 MG) cells were extracted using miRNAeasy (Qiagen™). The miRNAs from the 97 FFPE (formalin-fixed paraffin-embedded) surgically resected tumor specimens or the 8 non-tumoral brain tissues were extracted on 3 adjacent 15 μm cuts using the miRNAeasy-FFPE kit (Qiagen™, Hilden, Germany). For the tumoral specimens, morphological control was systematically carried out beforehand by a neuropathologist (ELZ) in order to guarantee that the percentage of tumor cells was greater than 70% and the absence of areas of necrosis or hemorrhage. When this was not the case, a macro-dissection of the samples was performed to determine these quality criteria. miRNAs were extracted from the seven plasma samples using NucleoSpin miRNA Plasma (Macherey-Nagel™, Düren, Luxembourg). For each sample, miRNA extraction was carried out according to the respective manufacturer’s instructions.
The integrity and quality of the purified DNA were assessed by 1% agarose gel electrophoresis, and the DNA/miRNA concentration was measured with the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Asnières-sur-Seine, France).
5
Quantification of miRNA and mRNA Levels
6
RNA Extraction and qPCR Analysis
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Total RNA was purified by miRNAeasy (Qiagen, Germantown, MD, USA) and reverse‐transcribed using TaqMan UNIVERSAL MMix II (Applied Biosystems, Waltham, MA, USA) for random priming or miRNA‐specific assay reverse transcription. Semi‐quantitative PCR was performed with TaqMan‐validated assays (Applied Biosystems). As endogenous reference gene for cDNA, we chose U6 (#001973) for miRNA. All analyses were carried out in triplicate. Real‐time data were collected using Microsoft Excel and analysed with the following formula: Expression level = 2‐ΔΔCt method.17 (link) All experiments were done as independent triplicates and analysed using standard deviation (SD). The p value was obtained with Student's t test.
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