For mRNA analysis, mRNA was isolated from cells using an Illustra RNA extraction kit (GE Healthcare, Ottawa, Canada) and reverse-transcribed using an iScript cDNA Synthesis Kit (Bio-Rad). iTaq Universal Probes Supermix (Bio-Rad) with PrimeTime Probes (IDT, Coralville, IA, USA) or SsoFast EvaGreen Supermix (Bio-Rad) with primers (Invitrogen) was used to run qPCR analysis on the 7500Fast system (Applied Biosystems, Foster city, CA, USA). For miRNA analysis, miRNA was isolated from cells using a miRNeasy miRNA isolation kit (Qiagen, Toronto, Canada) and TaqMan MicroRNA assays (Applied Biosystems) were performed according to manufacturer’s protocol to run qPCRs using the 7500Fast system. See Supp. Table 4 for miRNA assays and primers used for qPCR.
Mirneasy mirna isolation kit
Manufactured by Qiagen
Sourced in Canada, Spain, Germany
The MiRNeasy miRNA isolation kit is a product offered by Qiagen for the extraction and purification of microRNA (miRNA) from various sample types, including cells, tissues, and body fluids. The kit utilizes Qiagen's proprietary technology to efficiently isolate and concentrate miRNA molecules from the total RNA pool.
Automatically generated - may contain errors
Lab products found in correlation
Taqman microrna assay, by Thermo Fisher Scientific (2 mentions)
Illustra rna extraction kit, by GE Healthcare (1 mentions)
7500 fast system, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
Itaq universal probes supermix, by Bio-Rad (1 mentions)
Superscript 3 first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
Mirneasy serum plasma kit, by Qiagen (1 mentions)
Miscript reverse transcription kit, by Qiagen (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Viia7, by Thermo Fisher Scientific (1 mentions)
Quantstudio 12k flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Taqman pcr master mixture, by Thermo Fisher Scientific (1 mentions)
Taqman reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Taqman fast universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
6 protocols using mirneasy mirna isolation kit
1
Quantitative mRNA and miRNA Analysis
2
Fibroblast RNA Isolation and RT-PCR
3
Quantification of miR-155 in PBMC and Serum
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Total cellular RNA was isolated and purified from PBMCs or cell subsets using the miRNeasy miRNA isolation kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The cDNA synthesis was performed with 100 ng total miRNA using the miScript Reverse Transcription Kit (Qiagen). The expression of miR-155 genes was determined by real-time RT-PCR, which was performed with miScript SYBR Green PCR Kit (Qiagen) by using commercially available qPCR primer (Qiagen) on a LightCycler 480 (Roche Diagnostics International, Rotkreuz, Switzerland) according to the manufacturer’s instructions. The expression levels of each gene in the PBMCs were presented as values normalized against 106 copies of U6 small nuclear RNA (RNU6B) transcripts. The miRNA in serum was extracted by miRNeasy Serum/Plasma Kits (Qiagen) and was synthesized as above. In addition, C. elegans miR-39 was added to serum samples as an internal control according to manufacturer’s recommends. Then quantitative real-time RT-PCR analysis for each gene in serum was performed as it was for the PBMCs, and the results were presented as values normalized against 106 copies of C. elegans miR-39 miRNA transcripts. It was noteworthy that only 48 patients had enough corresponding serum retained to accomplish serum miR-155 detection.
4
RNA and MicroRNA Extraction and Quantification
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Total RNA and microRNAs were prepared from cells and tissues using the miRNeasy miRNA isolation kit (Qiagen) according to the manufacturer’s instructions. cDNA and miR cDNA synthesis were performed with high capacity RNA to cDNA kit and TaqMan MicroRNA Reverse Transcription kit respectively (Life Technologies). TaqMan assays were performed on a ViiA7 (Applied Biosystems) or a QuantStudio 12K Flex Real Time PCR System (Applied Biosystems) using the following conditions: 50°C for 2 minutes; 95°C for 10 minutes; 40 cycles of 95°C for 15 seconds; and 60°C for 1 minute. The expression of Ubiquitin was used for normalization of mRNA samples, whereas U6 was used for normalization of small RNA expression.
5
Quantification of miR-1827 and target genes
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The total RNA was purified by using a miRNeasy miRNA Isolation Kit (Qiagen) as we previously described [25 (link)]. The miR-1827 expression levels were determined by real-time PCR using Taqman primers and Taqman PCR master mixture (Applied Biosystems). The expression of miR-1827 was normalized with the expression of U6 snRNA. To detect the mRNA expression of MDM2 and p53 target genes, cDNA was prepared with random primers using TaqMan reverse transcription kit (Applied Biosystems) as previously described [33 (link), 53 (link)]. Gene expression levels were determined by real-time PCR using Taqman PCR master mixture and primers. The expression of genes was normalized to Actin gene.
6
Quantifying miR-155 and Ets1/Itk Gene Expression
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Fifty nanograms of total RNA, isolated by using the miRNeasy miRNA isolation kit (Qiagen), were subjected to reverse transcription according to the manufacturer’s instructions (Applied Biosystems). Quantitative Real time RT-PCR analysis for miR-155 (assay ID: 002571) was performed according to the TaqMan MicroRNA Assays (Applied Biosystems) and samples normalized by evaluating RNA U6 (assay ID: 001973) expression.
RNA was extracted according to the manufacturer’s instructions (RNeasy MICROKIT, Qiagen) and reverse transcribed using High-Capacity® cDNA Reverse Transcription Kits (Applied Biosystem). Real time RT-PCR were performed on 7900 HT (Applied Biosystem), using TaqMan® Fast Universal PCR masterMix (Applied Biosystem). Assays (Ets1 assay ID: Mm01175819_m1; Itk assay ID: Mm00439862_m1) and samples were normalized by evaluating HPRT1 (assay ID: Mm01545399_m1) expression. Results were obtained using the comparative Ct method.
RNA was extracted according to the manufacturer’s instructions (RNeasy MICROKIT, Qiagen) and reverse transcribed using High-Capacity® cDNA Reverse Transcription Kits (Applied Biosystem). Real time RT-PCR were performed on 7900 HT (Applied Biosystem), using TaqMan® Fast Universal PCR masterMix (Applied Biosystem). Assays (Ets1 assay ID: Mm01175819_m1; Itk assay ID: Mm00439862_m1) and samples were normalized by evaluating HPRT1 (assay ID: Mm01545399_m1) expression. Results were obtained using the comparative Ct method.
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