Mirnease mini kit

Manufactured by Qiagen

Sourced in Germany

The MiRNease Mini Kit is a laboratory equipment designed for the purification of small RNA molecules, including microRNA (miRNA), from various biological samples. The kit utilizes a silica-based membrane technology to efficiently capture and purify these small RNA species.

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Lab products found in correlation

Miscript sybr green pcr kit, by Qiagen (2 mentions) Lightcycler 480 sybr green 1 master, by Roche (2 mentions) Primescript rt reagent kit, by Takara Bio (1 mentions) Miscript 2 rt kit, by Qiagen (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions) Opti mem medium, by Thermo Fisher Scientific (1 mentions) Iscript reverse transcription supermix, by Bio-Rad (1 mentions) Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions) Dnase digestion, by Qiagen (1 mentions) Ebm 2 mv medium, by Lonza (1 mentions) Huvecs, by Lonza (1 mentions) Hiseq 4000, by Illumina (1 mentions) Poly t oligo attached magnetic beads, by Thermo Fisher Scientific (1 mentions) Mrna seq sample preparation kit, by Illumina (1 mentions) Miscript reverse transcription kit, by Qiagen (1 mentions) One step cell separation, by BD (1 mentions) Primescript rt kit, by Qiagen (1 mentions)

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Mini Kits (17 citations)

6 protocols using mirnease mini kit

Quantification of miRNA Expression

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Total RNA from the frozen samples or cultured cells was extracted using miRNease Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. PrimeScript RT reagent Kit (TaKaRa, Tokyo, Japan) was used to synthesize complementary DNA with500 ng of total RNA. qRT-PCRs were performed using 2μl of resulting cDNA per 20-μl reaction volume containing SYBR green I master. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6B small nuclear RNA were used as internal control. Controls without template DNA were negative. Real-time PCRs were performed on LightCycler 480 SYBR Green I Master (Roche, Welwyn Garden, Swiss). We incubated the reactions at 95℃ for 30 s followed by 40 cycles at 95℃ for 5s, at 55℃ for 10 s and at 72℃ for 15 s. To verify that the SYBR Green dye detected only one qRT-PCR product, we subjected the samples to the heat dissociation protocol after the final cycle of qRT-PCR to check for the presence of only peak. Mature miRNAs were quantified with specific primers using miScript II RT Kit (Qiagen) for reverse transcription and miScript SYBR Green PCR Kit (Qiagen) for subsequent qRT-PCR as specified by the manufacturer. The primers used in our study are listed in Table 2.

Sun Y., Xiao Y., Sun H., Zhao Z., Zhu J., Zhang L., Dong J., Han T., Jing Q., Zhou J, & Jing Z. (2019). miR-27a regulates vascular remodeling by targeting endothelial cells' apoptosis and interaction with vascular smooth muscle cells in aortic dissection. Theranostics, 9(25), 7961-7975.

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TGFβ2-induced gene expression in HUVECs

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HUVECs were purchased at Lonza and cultivated in EBM-2 MV medium (Lonza). siRNA transfections were done in OptiMem medium (ThermoFisher) with the lipofectamine RNAimax transfection reagent (ThermoFisher). siRNA was purchased from Qiagen (siRNA MAPK1_10 and MAPK3_6). TGFβ2 stimulation (R&D Systems) was done at 10 ng/ml in EBM-2 MV medium changed daily. RNA extractions were done using RNeasy mini kit (Qiagen) with DNase digestion (Qiagen). cDNA was synthetized with iScript Reverse Transcription Supermix (Bio-Rad), and qPCR was performed using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). For miRNA, RNA was isolated with miRNease Mini Kit (Qiagen), and cDNA and qPCR were realized using miScript PCR System and primers (Qiagen).

Ricard N., Scott R.P., Booth C.J., Velazquez H., Cilfone N.A., Baylon J.L., Gulcher J.R., Quaggin S.E., Chittenden T.W, & Simons M. (2019). Endothelial ERK1/2 signaling maintains integrity of the quiescent endothelium. The Journal of Experimental Medicine, 216(8), 1874-1890.

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Differential mRNA Expression in Blood

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The total RNAs in blood samples from patients before and after treatment for 3 days were extracted and purified by using EL buffer and miRNease Mini Kit from Qiagen (Germantown, MD) following the manufacture’s manual. RNA-sequencing was conducted by LC Sciences (Houston, TX). In their laboratory, total RNA was subjected to isolation of Poly(A) mRNA with poly-T oligo attached magnetic beads (Invitrogen, Carlsbad, CA). Following purification, the poly(A)- or poly(A)+ RNA fractions was fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library using mRNA-Seq Sample Preparation Kit (Illumina, San Diego). The average insert size for the paired-end libraries was 300 bp. Then the paired-end sequencing was performed on an Illumina Hiseq 4000 following the manufacturers protocol. Transcripts assembly and different expression mRNAs were conducted and analyzed. Gene set enrichment analysis was conducted using software GSEA and Molecular Signatures Database (Broad Institute, MA).

Seymour E.K., Yar Khan H., li Y., Chaker M., Muqbil I., Aboukameel A., Ramchandren R., Houde C., Sterbis G., Yang J., Bhutani D., Pregja S., Reichel K., Huddlestun A., Neveux C., Corona K., Landsman Y., Shah J., Kauffman M., Shacham S., Mohammad R.M., Azmi A.S, & Zonder J.A. (2021). Selinexor in combination with R-CHOP for frontline treatment of non-Hodgkin lymphoma: results of a phase 1 study. Clinical cancer research : an official journal of the American Association for Cancer Research, 27(12), 3307-3316.

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Quantification of miRNAs and lincRNA in hESCs

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RNA was extracted from hESCs (H1) with the miRNease Mini kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized from 1 μg of total RNA using the miScript Reverse Transcription kit (Qiagen). Quantification of miRNAs was performed using the miScript SYBR Green PCR kit (Qiagen) and the Hs-let-7a and Hs-let-7d miScript Primer Assays (Qiagen), according to the manufacturer’s instructions, on an ABI 7300 Real-Time PCR System. qRT-PCR was carried out using normalization to Hs-RNU6-2 as recommended. Differences in fold expression with regard to controls were calculated from triplicate C_t values following the 2^−ΔΔC_t method. HPAT5 lincRNA levels were quantified in parallel according to the manufacturer’s instructions. Experiments were carried out twice.

Durruthy-Durruthy J., Sebastiano V., Wossidlo M., Cepeda D., Cui J., Grow E.J., Davila J., Mall M., Wong W.H., Wysocka J., Au K.F, & Pera R.A. (2015). The primate-specific noncoding RNA HPAT5 regulates pluripotency during human preimplantation development and nuclear reprogramming. Nature genetics, 48(1), 44-52.

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PBMC Isolation from Whole Blood

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Blood was collected in cell preparation tubes (CPTs) designed for one-step cell separation (Becton Dickinson) from healthy controls and patients within 3 months of the date when the lung function tests were performed. The sample was immediately mixed and centrifuged at 1800 g at ambient temperature for 30 minutes. Total RNA from PBMC was extracted using the miRNease mini kit protocol (Qiagen). RNA samples were stored at –80 °C.

Christmann R.B., Wooten A., Sampaio-Barros P., Borges C.L., Carvalho C.R., Kairalla R.A., Feghali-Bostwick C., Ziemek J., Mei Y., Goummih S., Tan J., Alvarez D., Kass D.J., Rojas M., de Mattos T.L., Parra E., Stifano G., Capelozzi V.L., Simms R.W, & Lafyatis R. (2016). miR-155 in the progression of lung fibrosis in systemic sclerosis. Arthritis Research & Therapy, 18, 155.

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Quantitative RT-PCR for Gene Expression

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In the light of the manufacturer's instructions, total RNA was extracted from the serum using the miRNease Mini kit (Qiagen, Hilden, Germany), and 500 ng of total RNA was implemented to synthesize complementary DNA according to the PrimeScript RT kit (Qiagen). Based on these gene sequences published in GenBank, Primer 5.0 software was applied to design primers (Table 1), and checked by NCBI Blast and oligo 7. Then, LightCycler 480 SYBR Green I Master (Roche Diagnostics, Penzberg, Germany) was utilized for PCR reaction. Each SYBR Green reaction took 0.25 µL of primers and 2 µL of cDNA as templates, with a total volume of 20 µL. U6 and glyceraldehyde phosphate dehydrogenase were selected as internal references. 2 -△△Ct method was implemented to calculate the relative expression level.

Zhang Y., li J., Li J., Li-n M., & zhang S. (2020). Relationship between serum microRNA-223 and microRNA-126 and plaque stability in patients with carotid atherosclerosis.

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