Miscript sybr green

Manufactured by Qiagen

Sourced in Germany, United States

The MiScript SYBR Green is a laboratory equipment product by Qiagen. It is used for the detection and quantification of microRNA (miRNA) expression levels in various samples. The product employs the SYBR Green dye-based detection method to enable sensitive and reliable miRNA analysis.

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20 protocols using miscript sybr green

Quantifying miRNA Expression Levels

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miR levels were validated using miScript II RT kit (Qiagen), following manufacturer’s specifications for 5× miScript HiSpec Buffer. qPCR was performed using miScript SYBR Green (Qiagen) on CFX Connect (BioRad). Samples were assessed for expression of SNORD96A, miR-21a-5p, miR-223-3p, miR-29b-3p, miR-125a-5p, and miR-142-5p (Qiagen).

Sido J.M., Jackson A.R., Nagarkatti P.S, & Nagarkatti M. (2016). Marijuana-derived Δ-9-tetrahydrocannabinol suppresses Th1/Th17 cell-mediated delayed-type hypersensitivity through microRNA regulation. Journal of molecular medicine (Berlin, Germany), 94(9), 1039-1051.

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Quantitative Transcriptional Analysis

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Cells were harvested at 70–80% confluence in TRIzol (Invitrogen), and RNA was extracted per manufacturer’s instructions. RNA levels of specific transcripts were assessed by qRT-PCR (miScript SYBRgreen, Qiagen) with U6 RNA as the internal control (primers are listed in the Supplemental Methods).

Sechler M., Parrish J.K., Birks D.K, & Jedlicka P. (2017). The histone demethylase KDM3A, and its downstream target MCAM, promote Ewing Sarcoma cell migration and metastasis. Oncogene, 36(29), 4150-4160.

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Detecting Small RNA Contamination in Spin Columns

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RT-qPCR analysis was performed to assess contamination of spin columns with small RNAs and confirm findings from sequencing analysis. RNeasy MinElute spin columns (Qiagen) might be contaminated with small RNAs, thereby causing artifacts in microRNA analysis [14 (link)]. RNeasy MinElute columns were treated with sodium hypochlorite to remove possible contamination [14 (link)]. We dissolved a non-natural microRNA (miSpike; IDT DNA, Coralville, IA; 5’-rCrUrCrArGrGrArUrGrGrCrGrGrArGrCrGrGrUrCrU-3’) in 200 μL RNase-free, molecular biology grade water and extracted miSpike using MinElute columns. Eluted miSpike (100 ng) was reverse transcribed using the miScript II RT Kit (Qiagen). In addition, miR-30d-5p, miR-125a-5p and miR-423-5p were analyzed in hypochlorite-treated and untreated columns. RT-qPCR was performed using miScript SYBR Green (Qiagen), the universal reverse primer in the miScript II RT Kit and sequence-specific forward primers (Supplemental Table 3, Supplemental Digital Content 3). Hypochlorite-treated columns were used when confirming sequencing data by RT-qPCR. Melting curve analyses suggested formation of a single product (not shown). Ct values greater than 29 were considered not detectable [15 (link)].

Leiferman A., Shu J., Upadhyaya B., Cui J, & Zempleni J. (2019). Storage of Extracellular Vesicles in Human Milk, and MicroRNA Profiles in Human Milk Exosomes and Infant Formulas. Journal of pediatric gastroenterology and nutrition, 69(2), 235-238.

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Quantitative Transcriptional Analysis

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Quantitative analysis of miRNA and mRNA

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Total RNA, enriched for miRNAs and mRNA, was extracted using the miRNeasy Mini kit (Qiagen, Germany) and TransZol Up Plus RNA kit (TransGen, China) from whole insect bodies (n = 30), respectively. The miScript Ⅱ RT kit (QIAGEN) and the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen) were used to prepare cDNA. Quantitative real-time PCR (qPCR) reactions were conducted with an ABI Prism 7300 (Applied Biosystems, USA) using miScript SYBR Green (Qiagen, Germany) and SYBR® Premix Ex Taq II RR820A (Takara, Japan), according to the manufacturer’s instructions. The data was analyzed using the 2^-△△Ct method. Actin1 (GenBank accession No. EU179846.1) was used as the endogenous control. This gene has been studied to show it is suitable to be used as a reference gene [46 (link)]. All treatments were carried out in triplicate. Independent reactions were performed in quadruplicate for each RNA sample, and the signal intensity of the target gene is presented as the average value. The qPCR primers used in this study are listed in S3 Table.

Ye X., Xu L., Li X., He K., Hua H., Cao Z., Xu J., Ye W., Zhang J., Yuan Z, & Li F. (2019). miR-34 modulates wing polyphenism in planthopper. PLoS Genetics, 15(6), e1008235.

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Comprehensive RNA Profiling Protocol

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The purity of both the large and small RNA-enriched fractions were quantified on a nanodrop spectrometer, and further taken for cDNA conversion. The mature miRNA was selected and converted from a small RNA-enriched fraction using HiSpec buffer provided with the miScript II RT kit (Qiagen, USA). For lncRNA-cDNA, first-strand synthesis was carried out from a large RNA-enriched fraction using an RT2 first-strand kit (Qiagen, USA). The mRNA was converted to cDNA from a large RNA fraction using an oligo dT mixture provided with the kit (Bio-Rad, Hercules, CA, USA). To analyze the expression of the targets of the study, we used the respective primers as listed in Table 1. The lncRNA was quantitatively determined using RT2 SYBR green master mix (Qiagen, USA), and SYBR green super mix (Bio-Rad) was used to detect the target mRNAs. GAPDH served as an internal control for both lncRNA and mRNAs. With respect to miRNAs, miScript SYBR Green (Qiagen) was used with U6 as an internal control.

Architha T., Juanitaa G.R., Vijayalalitha R., Jayasuriya R., Athira G., Balamurugan R., Ganesan K, & Ramkumar K.M. (2024). LncRNA NEAT1/miR-146a-5p Axis Restores Normal Angiogenesis in Diabetic Foot Ulcers by Targeting mafG. Cells, 13(5), 456.

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Total RNA Extraction and qRT-PCR Analysis

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Total RNA was extracted from cells using miRNeasy (Qiagen, Valencia, CA) as previously described [57 (link)]. Equal amounts of RNA were reverse transcribed into cDNA using the miScript II RT Kit and qRT-PCR was conducted with miScript SYBR Green (Qiagen). mRNA expression was normalized to beta actin, while small RNA expression was normalized to RNU6B (RNU6B_13, Qiagen). The polyA site was identified in BCAR3 from PEO4 and 2008 cells using Thermoscript RT (Invitrogen) and oligo dT as previously described [58 ]. PCR products were amplified using the 5’ forward BCAR3 primer with the Universal reverse primer from Qiagen and amplified using GoTaq Green (Promega, Madison, WI). Primers are listed in Supplementary Table 3. PCR products were cloned into T-vector (Life Technologies, Carlsbad, CA) and sent for direct sequencing (University of Minnesota Genomic Center, Minneapolis, MN).

Zhou K., Diebel K.W., Holy J., Skildum A., Odean E., Hicks D.A., Schotl B., Abrahante J.E., Spillman M.A, & Bemis L.T. (2017). A tRNA fragment, tRF5-Glu, regulates BCAR3 expression and proliferation in ovarian cancer cells. Oncotarget, 8(56), 95377-95391.

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Validating miRNA Expression via qRT-PCR

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Real time qRT–PCR was conducted on all samples to validate the miRNA profiling results. Specific miScript Primer Assays for miR-29a, miR-29b and miR-29c (Qiagen, Valencia, CA) were used to verify the expression by q-PCR. Reverse transcriptase (RT) reactions were performed with miScript II RT kit (Qiagen, Valencia, CA). The reactions were in 20 μl total volume containing 100 ng total RNAs (including small RNAs), 1X miScript HiSpec buffer (1X miScript HiFlex buffer for RNU6B), 1X miScript Nucleics Mix, 1X miScript Reverse Transcriptase Mix. The reactions were incubated for 1 hour at 37°C, then 5 min at 95°C. All reverse transcriptase reactions were run in duplicate. Real-time PCR was performed on StepOnePlus Real-Time PCR System (Applied Biosystems). The PCR included the RT product, miScript SYBR Green and miScript Primer Assay (Qiagen, Valencia, CA). The reactions were incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative expression was determined using the ΔΔCT method. RNU6B was used as the internal control according to manufacturer instructions and consistent with prior studies.^{37 (link)}

Chen X., Talati M., Fessel J.P., Hemnes A.R., Gladson S., French J., Shay S., Trammel A., Phillips J.A., Hamid R., Cogan J.D., Dawson E.P., Womble K.E., Hedges L.K., Martinez E.G., Wheeler L.A., Loyd J.E., Majka S.J., West J, & Austin E.D. (2015). The Estrogen Metabolite 16αOHE Exacerbates BMPR2-Associated PAH Through miR-29-Mediated Modulation of Cellular Metabolism. Circulation, 133(1), 82-97.

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RNA Extraction and qPCR Analysis

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RNA was extracted from samples using QIAzol (Qiagen) following the manufacturer’s instructions. cDNA was synthesized using miScript II RT kit (Qiagen). Then, 1μl of a 1:5 diluted cDNA reaction was used in qPCR reactions over 40 cycles using miScript SYBR Green (Qiagen) according to the manufacturer’s protocol. Melt curve analysis was carried out at the end of each run. A list of all primers used in this study are provided in Supplementary Table 1.

Perdomo H.D., Hussain M., Parry R., Etebari K., Hedges L.M., Zhang G., Schulz B.L, & Asgari S. (2021). Human blood microRNA hsa-miR-21-5p induces vitellogenin in the mosquito Aedes aegypti. Communications Biology, 4, 856.

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Quantitative Analysis of miRNA and hY4 RNA

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Spike-in control cel-miR-39 mimic (miRNeasy Serum/Plasma Spike-In Control, Qiagen) was added to total RNA samples as internal control, and the mixed RNA was reverse-transcribed to cDNA using a reverse transcriptase kit (miScript II RT Kit, Qiagen). SYBR-based qRT-PCR was performed on an ABI7500 real-time PCR amplifier (Applied Biosystems) using specific primers (miScript primer Assay, Qiagen) and a universal PCR kit (miScript SYBR Green, Qiagen) according to the manufacturer’s protocol. For hY4 RNA fragments detection, U6 snRNA was used as internal control. Amplifying conditions were as follows: 95 °C for 15 min, followed by 40 cycles of 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 34 s [84 (link), 85 (link)]. Gene expression data were normalized to internal control expression, and the relative expression was determined as 2^{−Δ Ct}, where Δ Ct = Ct (target gene) − Ct (internal control).

Li C., Qin F., Hu F., Xu H., Sun G., Han G., Wang T, & Guo M. (2018). Characterization and selective incorporation of small non-coding RNAs in non-small cell lung cancer extracellular vesicles. Cell & Bioscience, 8, 2.

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