Fast sybr green

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Belgium

Fast SYBR Green is a real-time PCR reagent that enables rapid and sensitive detection of target DNA sequences. It utilizes the SYBR Green I dye, which binds to double-stranded DNA, to provide fluorescent signal detection during the PCR process.

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SYBR Green (3216 citations) Fast Green (27 citations) VeriQuest Fast SYBR Green qPCR Master Mix (14 citations) Fast SYBR Green Cells-to-Ct Kit (2 citations) Genious 2× SYBR Green Fast Qpcr Mix (2 citations) SYBR green fast reagent PCR master mix (2 citations) USB VeriQuest Fast SYBR Green qPCR Master Mix (2 citations) AB Fast SYBR Green Master Mix (2 citations) Fast SYBR Green detection (2 citations) Fast SYBR™ Green quantification (2 citations)

158 protocols using fast sybr green

qPCR Quantification of PREX2 Expression

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Quantitative polymerase chain reaction was performed in an ABI 7500 machine, using Fast SYBR Green (Invitrogen) according to the manufacturer’s instructions. The primer sequences were as follows: PREX2 forward, 5′-AACCATGAGAAGGCACAAAAA-3′, and reverse, 5′-CTTGCATATTCTTTGTATTGGTGT-3′. Samples were run in triplicate. The efficiencies of PREX2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were measured by using standard curves and were calculated to be 2.3 and 2.0, respectively. Quantitative analysis was performed on the basis of the ΔΔC_t method using the housekeeping gene GAPDH. The GAPDH primer sequences were as follows: forward, 5′-GAAGGTGAAGGTCGGAGTCAAC-3′, and reverse, 5′-CAGAGTTAAAAGCAGCCCTGGT-3′.

Mense S.M., Barrows D., Hodakoski C., Steinbach N., Schoenfeld D., Su W., Hopkins B.D., Su T., Fine B., Hibshoosh H, & Parsons R. (2015). PTEN inhibits PREX2-catalyzed activation of RAC1 to restrain tumor cell invasion. Science signaling, 8(370), ra32.

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Quantitative Real-Time PCR Assay

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Quantitative real‐time polymerase chain reaction (qPCR) was carried out using an RNeasy Micro Kit (Qiagen, Manchester, UK), SuperScript III Reverse Transcriptase (Invitrogen), and Fast SYBR Green (Invitrogen) and analyzed using an Applied Biosystems StepOnePlus Real‐Time PCR System. Table S3 lists the primers used. Gene expression was quantified from samples run in triplicate using the 2^‐(ΔΔCT) method,19 normalized to the mean of 3 endogenous control genes — β‐actin, GAPDH, and β2‐microglobulin — and then normalized to the mean of the healthy control samples.

Smith A.M., Depp C., Ryan B.J., Johnston G.I., Alegre‐Abarrategui J., Evetts S., Rolinski M., Baig F., Ruffmann C., Simon A.K., Hu M.T, & Wade‐Martins R. (2018). Mitochondrial dysfunction and increased glycolysis in prodromal and early Parkinson's blood cells. Movement Disorders, 33(10), 1580-1590.

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Quantitative Analysis of Gonadal Transcripts

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Total RNA was extracted from ovaries and testes using TRIzol™ (Invitrogen) following the manufacturer’s instructions. Total RNA (1 μg) was treated with DNase I at 37°C for 10 min before inactivating the enzyme at 70°C for 10 min with EDTA. cDNAs were synthesized using Superscript III reverse transcriptase (Invitrogen) following the manufacturer’s protocol. Each experiment was conducted in three biological replicates, with technical duplicates. Quantitative PCR was conducted with Fast SYBR™ Green (Invitrogen) and KAPA SYBR™ Fast (KAPA Biosystems) using StepOnePlus™ (Applied Biosystems, CA, United States). Relative expression levels were normalized to those of Actin5c, and fold change against heterozygous controls was compared. A list of primers is provided in Supplementary Table 1.

Lim L.X., Isshiki W., Iki T., Kawaguchi S, & Kai T. (2022). The Tudor Domain-Containing Protein, Kotsubu (CG9925), Localizes to the Nuage and Functions in piRNA Biogenesis in D. melanogaster. Frontiers in Molecular Biosciences, 9, 818302.

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Quantification of mRNA Expression via RT-PCR

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mRNA expression was quantitated using RT-PCR as previously described [5 (link),46 (link),47 (link),48 (link)]. Briefly, TRIzol (Life Technologies) extracted total RNA was quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific). Total RNA (250 ng) was subjected to cDNA synthesis using the High Efficiency cDNA Synthesis Kit (Life Technologies). The levels of various mRNA transcripts were determined using Fast SYBR Green (Life Technologies) and gene-specific forward and reverse primer sets (Table 1) on a ViiA7 instrument (Life Technologies). 18s RNA expression was used to normalize mRNA expression using the ΔΔCT comparative method.

Hamid T., Ismahil M.A., Bansal S.S., Patel B., Goel M., White C.R., Anantharamaiah G.M, & Prabhu S.D. (2020). The Apolipoprotein A-I Mimetic L-4F Attenuates Monocyte Activation and Adverse Cardiac Remodeling after Myocardial Infarction. International Journal of Molecular Sciences, 21(10), 3519.

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Total RNA Isolation and cDNA Synthesis

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Total RNA was isolated from primary CLL cells using the GenElute Mammalian Total RNA Miniprep Kit (Sigma) and cDNA was synthesized by reverse transcriptase reactions according to the manufacturer’s instructions (Promega). Products were amplified in a Fast SYBR green (Life Technologies) reaction (40 cycles of 5 s at 95 °C followed by 30 s at 60 °C).

Haselager M., Thijssen R., West C., Young L., Van Kampen R., Willmore E., Mackay S., Kater A, & Eldering E. (2021). Regulation of Bcl-XL by non-canonical NF-κB in the context of CD40-induced drug resistance in CLL. Cell Death and Differentiation, 28(5), 1658-1668.

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qRT-PCR Analysis of IK6 Expression

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RNA was prepared using spin columns (QIAGEN) and cDNA using SuperScript VILO (Life Technologies). Primers for qRT-PCR were designed to amplify across at least one intron and produce a product of 80–100 bp using Primer3 software (http://frodo.wi.mit.edu/). qRT-PCR reactions were performed using Fast SYBR green (Life Technologies) and a 7500 Fast Real-Time PCR analyzer (Applied Biosystems). Transcripts were normalized to ABL1 and/or B2M as indicated and the mean ΔCt values used to generate ratios of transcript levels in IK6 versus control cells.

Beer P.A., Knapp D.J., Kannan N., Miller P.H., Babovic S., Bulaeva E., Aghaeepour N., Rabu G., Rostamirad S., Shih K., Wei L, & Eaves C.J. (2014). A Dominant-Negative Isoform of IKAROS Expands Primitive Normal Human Hematopoietic Cells. Stem Cell Reports, 3(5), 841-857.

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Quantifying HTLV-1 Proviral Load

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Samples were donated by 29 ACs, 1 HTLV-1⁺ subject with polymyositis (P), 30 patients with HAM/TSP and 8 uninfected individuals (S1 Table). PBMCs were isolated from whole blood by density-gradient centrifugation using Histopaque-1077 (Sigma) and cryopreserved in FBS (Invitrogen) containing 10% dimethylsulphoxide (Sigma). Genomic DNA was extracted from unfixed PBMCs using the DNeasy kit (Qiagen) according to the manufacturer’s protocol. The lysis step was extended from 10 minutes at 56°C to 16 hours at 62°C when extracting DNA from formaldehyde-fixed cells. To quantify the PVL, a series of dilutions of genomic DNA starting from 5ng/μl was subjected to real-time quantitative PCR (qPCR) with the following primers: Tax: SK43 5’-CGGATACCCAGTCTACGTGT-3’ and SK44 5’-GAGCCGATAACGCGTCCATCG-3’; GAPDH: GAPDHF 5’- AACAGCGACACCCATCCTC-3’ and GAPDHR 5’- CATACCAGGAAATGAGCTTGACAA-3’. qPCR was performed using the QuantStudio 7 Flex real-time PCR system (Life technologies) with the standard Fast SYBRgreen (Life technologies) thermal cycle protocol. A patient-derived infected CD4⁺ T cell clone with a mapped single integrated provirus was used as a standard reference [41 (link)].

Manivannan K., Rowan A.G., Tanaka Y., Taylor G.P, & Bangham C.R. (2016). CADM1/TSLC1 Identifies HTLV-1-Infected Cells and Determines Their Susceptibility to CTL-Mediated Lysis. PLoS Pathogens, 12(4), e1005560.

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Quantitative PCR Analysis of CLL Markers

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Total RNA was isolated from primary CLL cells using the RNeasy Mini Kit (Qiagen) and cDNA was synthesized by reverse transcriptase reactions according to the manufacturer’s instructions (Promega). Products were amplified in a Fast SYBR green (Life Technologies) reaction (40 cycles of 5 s at 95 °C followed by 30 s at 60 °C). Primers used: CD40-Forward: TGATGTTGTCTGTGGTCCCC. CD40-Reverse: GGCAAACAGGATCCCGAAG. BCL-2-Forward: ATGTGTGTGGAGAGCGTCAA. BCL-2-Reverse: CAGTTCCACAAAGGCATCCCAG. BCL2L1-Forward (Bcl-XL): GTATTGGTGAGTCGGATCGC. BCL2L1-Reverse (Bcl-XL): TGCTGCATTGTTCCCATAGA.

Kielbassa K., Haselager M.V., Bax D.J., van Driel B.F., Dubois J., Levin M.D., Kersting S., Svanberg R., Niemann C.U., Kater A.P, & Eldering E. (2023). Ibrutinib sensitizes CLL cells to venetoclax by interrupting TLR9-induced CD40 upregulation and protein translation. Leukemia, 37(6), 1268-1276.

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Quantitative PCR Analysis of RNA

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Total RNA was extracted from HIE 96-well monolayers or MA104 cells grown to confluency in a 6-well plate using TRIzol reagent (Ambion). In RV-infection experiments, cells were harvested at 24 hpi. Total RNA was treated with Turbo DNase I (Ambion) and cDNA was generated from 250 ng RNA using the SensiFAST cDNA synthesis kit (Bioline). Quantitative PCR was performed using Fast SYBR Green (Life Technologies) with primers either designed in-house or published previously (72 (link)) (Table S3) and using the QuantStudio real time thermocycler (Applied Biosciences). Target genes were normalized to the housekeeping gene ribosomal subunit 18s and relative expression was calculated using the ddCT method. Experiments were performed three times independently, and data shown are representative of all replicates combined. For IL-1α, COX2, and iNOS experiments, HIE monolayers were Mock or RV (Ito)-infected and then treated with DMSO, 100U/mL apyrase, or 10 μM BPTU and normalized to 18s mRNA transcripts and relative to the Mock-DMSO condition.

Chang-Graham A.L., Perry J.L., Engevik M.A., Engevik K.A., Scribano F.J., Gebert J.T., Danhof H.A., Nelson J.C., Kellen J.S., Strtak A.C., Sastri N.P., Estes M.K., Britton R.A., Versalovic J, & Hyser J.M. (2020). Rotavirus induces intercellular calcium waves through ADP signaling. Science (New York, N.Y.), 370(6519), eabc3621.

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qPCR Analysis of Gene Expression

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Total RNA was isolated from cells using the miRNeasy kit (QIAGEN, 217004) and cDNA was synthesized from 1,000 ng total RNA using SuperScript IV First-Strand Synthesis SuperMix (Life Technologies, 18091200). qPCR was performed using Fast SYBRgreen (Life Technologies, 4385612) on a StepOnePlus Real-Time PCR system (Applied Biosystems). Relative expression was calculated using ΔΔCT values normalized against GAPDH expression. All primers were designed using primer 3 (http://frodo.wi.mit.edu/primer3/) and synthesized by Integrated DNA Technologies.

Deng Q., Natesan R., Cidre-Aranaz F., Arif S., Liu Y., Rasool R.U., Wang P., Mitchell-Velasquez E., Das C.K., Vinca E., Cramer Z., Grohar P.J., Chou M., Kumar-Sinha C., Weber K., Eisinger-Mathason T.S., Grillet N., Grünewald T, & Asangani I.A. (2022). Oncofusion-driven de novo enhancer assembly promotes malignancy in Ewing sarcoma via aberrant expression of the stereociliary protein LOXHD1. Cell reports, 39(11), 110971.

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