HEK293T cells were transfected using Lipofectamine 3000 (Invitrogen) with 255 ng gRNA expression plasmid (63.75 ng of plasmid DNA encoding each of four gRNAs targeting IL1RN or the off-target control HBG1) and 245 ng of pcDNA3.1-CRY-dCas9-VP64. Primer sequences and standard curves were previously described (20 (link)). IL1RN gRNAs were CR1 (TGTACTCTCTGAGGTGCTC, at –29), CR2 (ACGCAGATAAGAACCAGTT, at –180), CR3 (CATCAAGTCAGCCATCAGC, at –113), CR4 (GAGTCACCCTCCTGGAAAC). HBG1 control gRNAs were CR1 (GCTAGGGATGAAGAATAAA, –26), CR2 (TTGACCAATAGCCTTGACA, –101), (TGCAAATATCTGTCTGAAA, –163), (AAATTAGCAGTATCCTCTT, –209). As a negative control cells were transfected with 500 ng of an empty pCMV plasmid. Cells were either illuminated with blue light using a custom-built 6 × 4 LED array (1 s pulses every 15 s, 450 nm, 16 mW/cm2) starting 4 h after transfection or incubated in the dark for the entire experiment. Three days after transfection total mRNA was purified using Qiagen RNeasy spin prep columns. cDNA was synthesized using the SuperScript® VILO cDNA Synthesis Kit (Life Technologies). Relative levels of cDNA were detected using Quanta PerfeCta® SYBR® Green FastMix® (Quanta Biosciences) and CFX96 Real-Time PCR Detection System (Bio-Rad). Raw data was normalized to GAPDH levels and cells transfected with an empty plasmid control using the ΔΔCT method.
Quanta perfecta sybr green fastmix
Manufactured by Quanta Biosciences
Sourced in Germany
The Quanta PerfeCta SYBR Green FastMix is a ready-to-use reaction mix designed for quantitative real-time PCR (qPCR) applications. It contains all the necessary components, including SYBR Green I dye, required for the amplification and detection of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Taq pcr master mix kit, by Qiagen (2 mentions)
Qscript cdna supermix, by Quanta Biosciences (2 mentions)
Gelred nucleic acid gel stain, by Biotium (2 mentions)
Rneasy spin prep columns, by Qiagen (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Polytron pt 1200, by Kinematica (1 mentions)
Qtower3 thermocycler, by Analytik Jena (1 mentions)
3 protocols using quanta perfecta sybr green fastmix
1
CRISPR-Cas9 Modulation of IL1RN Gene
2
Quantification of Adrenergic Receptor Expression in sASCs
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Monolayer sASCs were lysed and pellets were homogenized mechanically (Polytron PT-1200, Kinematica). RNA isolation was performed using the NucleoSpin RNA kit (Machrey Nagel, Düren, Germany) according to the manufacturer’s instructions. cDNA synthesis was carried out using qScript cDNA Supermix (Quanta Biosciences, Beverly, MA, USA). Gene expression of α- (α1A, α1B, α1D, α2A, α2B, α2C), β-AR (β1, β2, β3) subtypes and TH was determined by qPCR using Taq PCR Master Mix kit (Qiagen, Hilden, Germany). The PCR products were run on a 1.8% (wt/vol) agarose gel, stained with GelRed Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA). Average score of AR and TH gene expression in sASCs from three different OA patients was calculated. In order to analyze the effects of NE and specific antagonists on chondrogenic differentiation of sASCs, real-time quantitative PCR was performed using Quanta PerfeCta SYBR Green FastMix (Quanta Biosciences) in a qTOWER3 real time PCR Thermocycler (Analaytik, Jena, Jena, Germany). Gene expression of chondrogenic markers (SOX9, COL2A1), of fibrous cartilage markers (COL1A1), and hypertrophic markers (RUNX2,COL10A1, and MMP13) was quantified. GAPDH served as endogenous control. Relative gene expression was determined using qPCR software (Analytic, Jena). All primers were synthesized by Thermo Fisher Scientific (Supplementary Table S1 ).
3
Gene Expression Analysis of Chondrocytes
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RNA isolation from monolayer chondrocyte pellets was performed using NucleoSpin RNA/Protein kit (Machrey Nagel, Düren, Germany) according to the manufacturer’s instructions. Synthesis of cDNA was performed using qScript cDNA Supermix (Quanta Biosciences, VWR, Darmstadt, Germany). For gene expression analysis of different AR subtypes and the IL-1β receptor (IL-1βR) on chondrocytes at day 0 and day 7, reverse transcription PCR was used (Taq PCR Master Mix kit, Qiagen, Hilden, Germany). PCR products were run on a 1.8% (wt/vol) agarose gel, stained with GelRed Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA). In addition, TH gene expression was quantified in order to consider possible autocrine effects. GAPDH served as housekeeping gene. In addition, gene expression changes of ARs and ECM-related genes (SOX9; COL1A1; COL2A1; COL10A1; COMP, ACAN; MMP13; ADAMTS-4; ADAMTS-5) after seven days of monolayer culture were analyzed using Quanta PerfeCta SYBR Green FastMix (Quanta Biosciences, VWR, Darmstadt, Germany) in qTOWER3 Thermocycler (Analytik Jena, Jena, Germany). Relative gene expression was determined by the ∆∆Ct method using qPCR3.2 software (Analytik Jena) [52 ]. Human RPII served as housekeeping gene [53 (link),54 (link)]. All primers were synthesized by Thermo Fisher Scientific (Table 2 ).
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