Mirna extraction kit

Manufactured by Qiagen

Sourced in United States, Germany

The MiRNA extraction kit is a laboratory tool designed for the isolation and purification of microRNA (miRNA) from various biological samples, such as cells, tissues, and biofluids. The kit utilizes a selective and efficient extraction process to capture and concentrate miRNA molecules, enabling their subsequent analysis and study.

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MiRNA extraction kit and qPCR probes for specific miRNAs RNA and miRNA extraction kits MiRNeasy Serum/Plasma miRNA extraction kit Allprep DNA/RNA/miRNA universal extraction kit AllPrep DNA/RNA/miRNA extraction kit MiRNeasy miRNA extraction kit

11 protocols using mirna extraction kit

Quantification of miR-21 in Urine Samples

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A serial dilution series of miR-21 between 10⁻⁸ and 10⁻¹⁴ M was prepared in TMD buffer. As we have described in detail elsewhere [7] , RT-qPCR was then carried out on these dilutions directly, or following extraction using a Qiagen^® miRNA extraction kit. Aliquots from the 5 urine samples described above were processed using the Qiagen^® miRNA extraction kit. Where appropriate extracts were frozen at −80 °C prior to analysis using standard RT-qPCR protocols (vide supra).

Smith D.A., Newbury L.J., Drago G., Bowen T, & Redman J.E. (2017). Electrochemical detection of urinary microRNAs via sulfonamide-bound antisense hybridisation. Sensors and Actuators. B, Chemical, 253, 335-341.

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Temporal Cortex miRNA and HIF-1α in mTLE

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Temporal cortex tissues resected from 22 patients with refractory mTLE who had undergone anterior temporal lobectomy were collected. The 20 control samples of temporal neocortical tissues without abnormal pathological changes were obtained. None of patients in control group was ever diagnosed of epilepsy or seizures. Resected brain tissues were immediately frozen in liquid nitrogen for further studies.
For miRNA expression analysis, small RNAs from temporal cortex tissue from patients or controls were isolated using miRNA extraction kit (Qiagen) according to manufacture’s instruction. For detection of HIF-1α mRNA in brain tissue, RNA was extracted according to Trizol method (Invitrogen, USA).

Gong G.H., An F.M., Wang Y., Bian M., Wang D, & Wei C.X. (2018). MiR-153 regulates expression of hypoxia-inducible factor-1α in refractory epilepsy. Oncotarget, 9(9), 8542-8547.

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miRNA extraction from plasma and tumor tissue

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The plasma (200 µl) and tumor tissue (5 mg) samples
were subjected to miRNA extraction. Frozen plasma
samples were thawed and miRNAs were extracted
using a miRNA extraction kit (Qiagen, Valencia, CA,
USA) according to the manufacturer’s instructions.
Tissue miRNAs (tumor and the corresponding
normal tissues) were isolated by a modified TRIzol
protocol as explained previously (14 (link)). The quantity
and quality of the extracted RNA was evaluated
using spectrophotometry and gel electrophoresis,
respectively.

Eslamizadeh S., Heidari M., Agah S., Faghihloo E., Ghazi H., Mirzaei A, & Akbari A. (2018). The Role of MicroRNA Signature as Diagnostic Biomarkers in Different Clinical Stages of Colorectal Cancer. Cell Journal (Yakhteh), 20(2), 220-230.

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Quantitative Gene Expression Analysis

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Total RNA was extracted from cells and tissues using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using the Primer-Script™ One Step RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China). qPCR was subsequently performed using SYBR^® Premix Ex Taq (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. The following thermocycling conditions were used: 95°C for 5 min; 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 20 sec. Differences in the expression of Bcl-2-associated X (Bax), B-cell lymphoma-2 (Bcl-2), caspase-3 and mammalian target of rapamycin (mTOR) in the four groups were compared. The relative mRNA expression levels were quantified using the 2^−ΔΔCq method (26 (link)) and normalized to the internal control GAPDH.
For the detection of miRNA, total RNA was isolated using the miRNA Extraction kit (Qiagen GmbH, Hilden, Germany). Total RNA was reverse transcribed into cDNA using the miRNA cDNA Synthesis kit (Qiagen GmbH). qPCR was subsequently performed using the SYBR^® Green (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. The experiment was performed as described above. U6 served as an internal control. The sequences of the primers utilized are listed in Table I.

Yao H., Sun Q, & Zhu J. (2019). miR-1271 enhances the sensitivity of colorectal cancer cells to cisplatin. Experimental and Therapeutic Medicine, 17(6), 4363-4370.

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Temporal Cortex Tissue Analysis in mTLE

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Temporal cortex tissues resected from 32 patients with refractory mTLE who had undergone anterior temporal lobectomy were collected. The 18 control samples of temporal neocortical tissues without abnormal pathological changes were obtained from neurosurgery department of the same hospital. The control group included 5 head trauma and 13 cerebral hemorrhage cases. None of patients in control group was ever diagnosed of epilepsy or seizures. Resected brain tissues were immediately minced to small pieces and frozen in liquid nitrogen for further studies. For miRNA expression analysis, small RNAs from temporal cortex tissue from patients or controls were isolated using miRNA extraction kit (Qiagen) according to manufacture’s instruction. For detection of HIF-1α mRNA in brain tissue, RNA was extracted according to Trizol method (Invitrogen, USA).

Li Y., Huang C., Feng P., Jiang Y., Wang W., Zhou D, & Chen L. (2016). Aberrant expression of miR-153 is associated with overexpression of hypoxia-inducible factor-1α in refractory epilepsy. Scientific Reports, 6, 32091.

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miRNA Extraction and Amplification

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The main experimental reagents were as follows: (1) RPMI1640 (provided by Gibco, USA); (2) DEME (provided by Gibco, USA); (3) fetal calf serum (provided by Gibco, USA); (4) a miRNA extraction kit (provided by Qiagen, USA); (5) a reverse transcription and amplification kit (provided by Qiagen, USA); and (6) 0.25% trypsin (provided by Gibco, USA).

Li L.Z., Wu Z.Z, & Lv Z. (2021). The Clinical Significance of miR-21 in Guiding Chemotherapy for Patients with Osteosarcoma. Pharmacogenomics and Personalized Medicine, 14, 1247-1261.

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Extraction of Total RNAs from Cell and Tissue Samples

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According to the manufacturer’s instruction, the total RNAs of 1 mL cell solution (1 × 10⁶ cells/mL) were extracted by the commercially available miRNA extraction kit (Qiagen Co., Inc., New York, NY, USA) in the ultra-clean platform. The extracted total RNAs were dispersed in 50 μL DEPC water. Thirty milligrams (30 mg) of thyroid pathological tissue samples were crushed and homogenized. The total RNAs of pre-treated tissue sample were extracted by the miRNA extraction kit in ultra-clean platform and re-dispersed in 50 μL DEPC water.

Wu Y., Gao J., Wei J., Zhou J., Meng X, & Wang Z. (2021). Development of Flow Cytometric Assay for Detecting Papillary Thyroid Carcinoma Related hsa-miR-146b-5p through Toehold-Mediated Strand Displacement Reaction on Magnetic Beads. Molecules, 26(6), 1628.

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Komagataella phaffii Small RNA Profiling

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Wild type Komagataella phaffii strain NRRL Y-11430 was cultivated in 5 mL of rich defined medium (RDM)^{45 (link)} with 4% glycerol by volume. Five identical cultures independently cultivated were used as biological replicates. Cultures were inoculated at OD600 = 0.1, and grown at 30°C for 24 hours until OD600 ≈ 16. Total RNA was extracted using the Qiagen miRNA extraction kit. RNA was converted to cDNA libraries using the New England Biolabs Small RNA Kit and sequenced on an Illumina NextSeq. Reads were aligned to the K. phaffii genome⁴⁶ using Salmon.^{47 (link)} Small RNAs were defined as continuous regions of >50 read depth. Expression of each small RNA was calculated using transcripts per million (tpm), normalized by sample.
To quantify edge resolution of each small RNA, we defined each edge of the RNA as the 20 bp upstream and downstream of the annotated boundary. Over this region, we calculated the change in read depth at each base pair. We defined edge resolution as the ratio of the maximum change in read depth to the maximum read depth.

Edge resolution = \frac{max (Δ read depth)}{max (read depth)}

The edge resolution score was then calculated as the mean of the 5’ and 3’ edge resolution for each small RNA. An edge resolution of 1 indicates that all paired end reads start and end at the same 5’ and 3’ base pairs.

Dalvie N.C., Leal J., Whittaker C.A., Yang Y., Brady J.R., Love K.R, & Love J.C. (2019). Host-informed expression of CRISPR guide RNA for genomic engineering in Komagataella phaffii. ACS synthetic biology, 9(1), 26-35.

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Quantitative Analysis of miR-34a in ESCC

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Total RNA was isolated from 78 cases of ESCC tissues, 25 cases of non-tumor tissues, and ESCC cell lines transfected with miR-34a mimic, inhibitor, si-PLCE1, and their own blank vector using a miRNA Extraction Kit (Qiagen, Hilden, Germany). Among the tissue samples, there were 25 paired ESCC tissues and their corresponding non-tumor tissues were selected randomly. Reverse transcription for the quantification of miRNAs was conducted via miRNA cDNA Synthesis Kit (Qiagen). The qPCR amplification of miR-34a was executed via SYBR green Premix Ex Taq II (Qiagen) using Step One Plus Real-Time PCR System (Applied Biosystems). The expression level of miRNA was normalized using U6 as an internal control. The experiment was executed in ABI Prism 7500 Sequence Detection System. The reaction was carried out with an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The expression level of miR-34a gene in all cases was calculated by 2^−ΔCt method, ΔCt = Ct miR-34a - Ct U6. The difference of miR-34a expression in ESCC and corresponding normal tissues was represented by the RQ value, which was 2^−ΔΔCt (ΔΔCt = ΔCt ESCC-ΔCt corresponding normal tissues), indicating the significant miR-34a gene expression of ESCC in tissues compared with adjacent normal tissue. Primers were used as follows:
MiR-34a: 5′-CCCAGAACATAGACACGCTGGA-3′
U6: 5′-TGGTGAAGACGCCAGTGGA-3′

Cui X.B., Peng H., Li R.R., Mu J.Q., Yang L., Li N., Liu C.X., Hu J.M., Li S.G., Wei Y., L.a.i.b.o.-.Y.i.n., Zhou H., Li F, & Chen Y.Z. (2017). MicroRNA-34a functions as a tumor suppressor by directly targeting oncogenic PLCE1 in Kazakh esophageal squamous cell carcinoma. Oncotarget, 8(54), 92454-92469.

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RNA Extraction and Microarray Analysis

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Total RNA was extracted using miRNA extraction kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. Microarray analysis was conducted in the NHGRI-NINDS-NIMH Microarray Core (Abdel Elkahloun, Director; NHGRI, Rockville, MD, USA) using its established protocols and employing RNA samples with a RIN (ie, RNA integrity number) of eight or higher and Clariom-S rat microarray chips (Affymetrix, Santa Clara, CA, USA), with post-hybridisation, normalisation and statistical analysis as described previously.^{26 (link)} Partek Genomic Suite software (Partek Inc., St Louis, MO, USA) was used to determine the differential expression of genes. The original CEL data files for all microarrays are available upon request.

Xu W., Dahlke S.P., Emery A.C., Sung M., Chepurny O.G., Holz G.G, & Eiden L.E. (2021). Cyclic AMP-dependent activation of ERK via GLP-1 receptor signalling requires the neuroendocrine cell-specific guanine nucleotide exchanger NCS-RapGEF2. Journal of neuroendocrinology, 33(7), e12974.

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