Sybr green

Manufactured by Qiagen

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SYBR Green is a fluorescent dye used in real-time PCR (polymerase chain reaction) applications. It binds to double-stranded DNA, emitting a fluorescent signal that can be detected and quantified during the PCR process. The intensity of the fluorescence is proportional to the amount of DNA present, allowing for the quantification of target DNA sequences.

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SYBR Green/ROX master mix (24 citations) RT2 SYBR Green Fluor qPCR Mastermix (20 citations) QuantTect® SYBR® Green RT-PCR Kit (5 citations) SYBR Green real-time RT-PCR (3 citations) MiRCURY LNA TM EXILENT SYBR Green (3 citations) RT2first SYBR green/ROX PCR master mix (3 citations) RT² SYBR Green FAST Mastermix (3 citations) Hot-start SYBR Green PCR kit (2 citations) SYBR Green I method (2 citations) QuantiTect One-Step SYBR-Green qRT–PCR (2 citations)

372 protocols using sybr green

Quantitative Analysis of Target mRNA Expression

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The expression level of target messenger RNA (mRNA) was analyzed by real-time quantitative PCR. TRIZOL reagent (Sigma-Aldrich) and RNeasy Mini Kit (Sigma-Aldrich) were used to extract total RNA from samples. Complementary DNA (cDNA) was synthesized with Omniscript reverse transcription (RT) kit (QIAGEN, Hilden, Germany) and qPCR was amplified with SYBR Green (QIAGEN). The CFX96 system (Bio-Rad Laboratories, Hercules, CA, USA) was used for quantitative analysis of RT-qPCR gene expression. Each real-time PCR sample (10 µL) contained 1 µL cDNA and 5 µL SYBR Green (QIAGEN), and the final concentration of forward and reverse primers was 10 µM.
All reactions were carried out at 95 °C for 10 minutes, then denatured at 95 °C for 2 seconds, annealed at 60 °C for 20 seconds, and extended at 70 °C for 10 seconds. Forty cycles were performed. A melt curve was produced as temperature was increased from 70 °C to 95 °C in 0.5 °C increments within 5 seconds to ensure specificity of amplification. Taking GAPDH as reference, the relative expression level was calculated using the ΔΔCt method. The primer sequences are shown in Table 1.

Che F., Han Y., Fu J., Wang N., Jia Y., Wang K, & Ge J. (2021). LncRNA MALAT1 induced by hyperglycemia promotes microvascular endothelial cell apoptosis through activation of the miR-7641/TPR axis to exacerbate neurologic damage caused by cerebral small vessel disease. Annals of Translational Medicine, 9(24), 1762.

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Quantification of mRNA Expression

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Analysis of mRNA/µg in muscle was done using TaqMan reverse transcription reagents (Life Technologies Waltham, MA, USA) or the fluorescent intercalating DNA SYBR Green (Qiagen, Hilden, Germany). GUSB (β-Glucuronidase) or TBP (Tata binding protein) or IPO8 (Importin 8) were used as housekeeping genes. Genes analyzed in this study and their related sequences are listed in Supplementary Table S1. To normalize for three housekeeping genes, we used the ratio between the arithmetic average and the geometric average of the ng of cDNA of each gene, as described in [78 (link)]. We loaded each well of 96-well plates with 20 ng of cDNA (2 μL) obtained with retro-transcription, supplemented with either the primers and the SYBR Green (Qiagen, Hilden, Germany) mix or the probe and the TaqMan Mix (ThermoFisher Scientific, Waltham, MA, USA). In both cases, water was added to a volume of 11 μL. The PCR cycle for Real-Time PCR was as follows: step 1, 95 °C for 15 min; step 2, 95 °C for 25 sec; step 3, 60 °C for 1 min; repeating steps 2 and 3 for 40 cycles. The instrument used for these assays is a 7900HT Fast Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA).

Re Cecconi A.D., Forti M., Chiappa M., Zhu Z., Zingman L.V., Cervo L., Beltrame L., Marchini S, & Piccirillo R. (2019). Musclin, A Myokine Induced by Aerobic Exercise, Retards Muscle Atrophy During Cancer Cachexia in Mice. Cancers, 11(10), 1541.

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Quantitative PCR Analysis of NSCLC Cells

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For qRT-PCR, NSCLC cells were cultured at subconfluency, RNA was isolated by Trizol extraction and reverse transcribed using MMLV reverse transcriptase (Invitrogen) and random primers, and PCR was performed in triplicate using SYBR green (Qiagen, Bio-Rad) and specific primers for each gene (Supplemental Table 1) in a 7900HT Fast Real-Time PCR machine (Applied Biosystems). Results were computed relative to a standard curve made with cDNA pooled from all samples and normalized to β-Actin. For qPCR of mitochondrial DNA, NSCLC cells were cultured at subconfluency, cells were lysed in RIPA buffer with Proteinase K, and DNA was purified with phenol/chloroform extraction followed by ethanol precipitation. PCR was performed in triplicate using SYBR green (Qiagen, Bio-Rad) and mouse-specific primers for each mitochondrial gene. Results were computed relative to a standard curve made with DNA pooled from all samples and normalized to β2-microglobulin.

Van Nostrand J.L., Brisac A., Mello S.S., Jacobs S.B., Luong R, & Attardi L.D. (2015). The p53 Target Gene Siva Enables Non-Small Cell Lung Cancer Development. Cancer discovery, 5(6), 622-635.

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Quantifying EGF Signaling Transcripts

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Measurement of changes in mRNA levels of 84 EGF signaling related genes were achieved with the RT² Profiler^™ PCR Array Human EGF / PDGF Signaling Pathway Kit (Cat.PAHS-040Z, Qiagen). Total RNA was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Then cDNA was synthesized from 2.0 μg mRNA using Superscript III first strand synthesis system (Thermo Fisher Scientific, Waltham, MA). SYBR green (Qiagen) was used to amplify cDNA and then cDNA was subjected to quantitative real time PCR, using SYBR green labeling, as directed by the manufacturer. Expression of EGF regulated transcripts was compared between the groups.

Wang W., Yang X., Dai J., Lu Y., Zhang J, & Keller E.T. (2019). Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion. Oncogene, 38(23), 4540-4559.

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Quantifying Serum miRNA Profiles by RT-qPCR

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Serum miRNAs were detected and quantified by Real-Time PCR using Universal RT miRNA PCR System (Exiqon). 4 μl of the eluted RNA was used as template for retrotranscription in a final volume of 20 μl. cDNA was diluted 1/11 with nuclease-free sterile water and 4 μl was used as template for PCR reaction.
Real time PCR detection was performed using SYBR Green and specific commercially available probes (Exiqon) for each miRNA of interest. Master Mix preparation and temperature cycles were performed following manufacturer’s instructions. All reactions were carried out in triplicates in Light Cycler 480 equipment (Roche) and Cq values were calculated using 2^nd derivative method (Light Cycler 480 Software 1.5, Roche). miRNA expression values are expressed as ΔCq, obtained from the following formula: ΔCq = miRNA Cq—Spike In Cq.
As previously mentioned, miRNA stability after few freeze/thawing cycles was checked in our lab demonstrating high stability, as previously reported [22 (link)].

Aguado-Fraile E., Ramos E., Conde E., Rodríguez M., Martín-Gómez L., Lietor A., Candela Á., Ponte B., Liaño F, & García-Bermejo M.L. (2015). A Pilot Study Identifying a Set of microRNAs As Precise Diagnostic Biomarkers of Acute Kidney Injury. PLoS ONE, 10(6), e0127175.

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Exosomal miRNA Profiling in Urine

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Exosomal samples and UBtip cells were lysed in Qiazol (Qiagen), extracted with Chloroform (Sigma) and purified using miniElute columns (Qiagen). The RNA was eluted into nuclease-free water and stored at −70°C until needed. The RNA concentration, quality and size were measured using picoRNA Kit BioAnalyser (Agilent) and followed by cDNA synthesis (Exiqon). cDNA was diluted 40× and used in a custom-designed qPCR miRNA plate using SybrGreen (Exiqon). The PCR was performed on a BioRad CFX96 machine, and we followed specific protocols for the chosen primer qPCR set-up. The miRNA targets were chosen based on their role in the Wnt/β-catenin pathway [32] and on sequencing data (high number of repeats) of exosomal samples derived from human urine [33].

Krause M., Rak-Raszewska A., Naillat F., Saarela U., Schmidt C., Ronkainen V.P., Bart G., Ylä-Herttuala S, & Vainio S.J. (2018). Exosomes as secondary inductive signals involved in kidney organogenesis. Journal of Extracellular Vesicles, 7(1), 1422675.

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Serum microRNA Profiling Protocol

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Serum samples were coded and blinded and RNA was extracted from 250 μl of serum with miRNeasy (Qiagen, Valencia, CA). RNA (4 μl) was reverse transcribed using the Universal cDNA Synthesis Kit (Exiqon Inc., Denmark). Exogenous oligonucleotides were spiked into the samples prior to each step to control for variation in RNA extraction (cel-miR-39), cDNA synthesis (Sp6), and inter-plate calibration (Sp3). qPCR was run on a ViiA 7 Real-Time PCR System (Applied Biosystems, Foster City, CA) using Exiqon SYBR green and custom Pick&Mix miRNA PCR plates containing primers for 21 miRNAs of interest and the controls Sp3, Sp6, and cel-miR-39 (Exiqon, Inc). Wells with Ct>37 or poor melting curves were excluded from analysis.

Mihelich B.L., Maranville J.C., Nolley R., Peehl D.M, & Nonn L. (2015). Elevated Serum MicroRNA Levels Associate with Absence of High-Grade Prostate Cancer in a Retrospective Cohort. PLoS ONE, 10(4), e0124245.

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Profiling microRNA in Ox-LDL Treated Endothelial Cells

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For analysis of microRNA in endothelial cells, RNA was isolated from H5V murine endothelial cells after treatment with ox-LDL (50 μg/ml) for 1 h, 6 h or 24 h. Total RNA was extracted with a phenol/chloroform EZRNA kit (Biological Industries, Beit Ha-Emek, Israel). For miRNA detection, 25 ng of total RNA was transcribed to cDNA using the universal RT LNATM cDNA Synthesis Kit (Exiqon, Vedbaek, Denmark). Quantitative real-time PCR was performed with Sybr Green and specific locked nucleic acid primers for miR-106b, miR-93 and miR-25 (Exiqon, Denmark), using a StepOnePlus instrument (Applied Biosystems). Results were derived by the Comparative CT (ΔΔCt) method and were normalized to the expression of U6.

Semo J., Chernin G., Jonas M., Shimoni S, & George J. (2019). Deletion of the Mir-106b~ 25 MicroRNA cluster attenuates atherosclerosis in Apolipoprotein E knockout mice. Lipids in Health and Disease, 18, 208.

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Quantification of miRNA Expression

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For primary miRNA expression, RNA was reverse transcribed with a high‐capacity cDNA archive kit (Life Technologies), and RT–qPCR performed using SYBR Green (Promega). The utilized primers are listed in Table EV1. For mature miRNA expression, RNA was reverse transcribed using miRCURY LNA Universal RT (Qiagen). SYBR Green was used to perform the RT–qPCR for mature miR‐143, miR‐145, using U6 as internal control (Exiqon).

Hall I.F., Climent M., Viviani Anselmi C., Papa L., Tragante V., Lambroia L., Farina F.M., Kleber M.E., März W., Biguori C., Condorelli G, & Elia L. (2021). rs41291957 controls miR‐143 and miR‐145 expression and impacts coronary artery disease risk. EMBO Molecular Medicine, 13(10), e14060.

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Single-Cell Genomic and Transcriptomic Profiling

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Genomic DNA and total RNA from 10 cells underwent physical isolation by using hypotonic lysis followed by magnetic separation. For the quantification of isolated genomic DNA, the LINE-1 locus was amplified by real-time PCR using SYBR Green (Exiqon) according to the manufacturer's protocols. Fractionated total RNA was used as a template for cDNA synthesis with a Single Cell-to-CT Kit (Life Technologies). Detailed validation methods are described in the Supplemental Methods.

Han K.Y., Kim K.T., Joung J.G., Son D.S., Kim Y.J., Jo A., Jeon H.J., Moon H.S., Yoo C.E., Chung W., Eum H.H., Kim S., Kim H.K., Lee J.E., Ahn M.J., Lee H.O., Park D, & Park W.Y. (2018). SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells. Genome Research, 28(1), 75-87.

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