End-point PCR amplification was performed using JumpStart RedTaq (Sigma). The Applied Biosystems Veriti 96-well thermal cycler (Applied Biosystems) and Gel DOC EZ imager (Bio-Rad) were used analyses. Gene expression was quantified by real-time qPCR in CFX96 Normal-Well Real-Time System (BioRad) using PerfeCta SYBR Green FastMix and specific oligonucleotide primers. The reaction mixtures contained 10 μl PerfeCta SYBR Green FastMix, 7.2 μl ddH2O, 2.0 μl template cDNA and 0.4 μl gene-specific 10 μM PCR oligonucleotides primers. The reaction conditions were 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s and Melt Curve (dissociation stage). Relative gene expression was calculated as delta (Δ Re (the difference between the cycle threshold values, Ct, of the internal control, and Ct of gene of interest) and confirmed by 2–ΔΔ CT method.
Perfecta sybr green fastmix
Manufactured by Bio-Rad
PerfeCTa SYBR Green FastMix is a pre-formulated qPCR reagent that contains all the necessary components for real-time PCR amplification and detection using SYBR Green I dye chemistry. It is designed to provide rapid, sensitive, and reproducible quantification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (4 mentions)
High capacity rna to cdna master mix, by Thermo Fisher Scientific (2 mentions)
Precellys lysing kit, by Bertin Technologies (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Quick rna miniprep plus kit, by Zymo Research (2 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (2 mentions)
Jumpstart redtaq, by Merck Group (1 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
Gel doc ez imager, by Bio-Rad (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
4 protocols using perfecta sybr green fastmix
1
Quantitative gene expression analysis by real-time qPCR
2
Quantitative Gene Expression Analysis via qPCR
3
Quantitative Gene Expression Analysis via qPCR
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For cells cultured in vitro, RNA isolation was performed with the Quick-RNA MiniPrep Plus Kit (Zymo). Reverse transcription was performed using the High Capacity RNA-to-cDNA Master Mix (Applied Biosystems), and qPCR was performed using PerfeCTa SYBR Green FastMix (Quanta) in a 7500 Real time PCR system (Applied Biosystems).
The xenograft tumours were snap-frozen with liquid nitrogen immediately after resection, and homogenized with Precellys Lysing kits (Bertin Instruments). Next, total RNA was extracted using standard TRIzol RNA extraction protocol. The cDNA was synthesized with SuperScript III First-Strand Synthesis System (Thermo Fisher), and qPCR was performed using PerfeCTa SYBR Green FastMix in a CFX Connect Real-Time PCR Detection System (BioRad).
Samples were amplified by 40 cycles of 10 seconds at 95°C and 30 seconds at 60°C. Results were calculated by the change-in-cycling-threshold (ΔΔCt) method as follows (relative to the reference control gene B2M and Gapdh, encoding β-2-microglobulin and glyceraldehyde phosphate dehydrogenase, respectively): −ΔΔCt = −(ΔCt Treatment − ΔCt Control), where ΔCt = Ct Target − ΔCt Ref.
The sequences of primers used can be found inSupplementary Table 6 .
The xenograft tumours were snap-frozen with liquid nitrogen immediately after resection, and homogenized with Precellys Lysing kits (Bertin Instruments). Next, total RNA was extracted using standard TRIzol RNA extraction protocol. The cDNA was synthesized with SuperScript III First-Strand Synthesis System (Thermo Fisher), and qPCR was performed using PerfeCTa SYBR Green FastMix in a CFX Connect Real-Time PCR Detection System (BioRad).
Samples were amplified by 40 cycles of 10 seconds at 95°C and 30 seconds at 60°C. Results were calculated by the change-in-cycling-threshold (ΔΔCt) method as follows (relative to the reference control gene B2M and Gapdh, encoding β-2-microglobulin and glyceraldehyde phosphate dehydrogenase, respectively): −ΔΔCt = −(ΔCt Treatment − ΔCt Control), where ΔCt = Ct Target − ΔCt Ref.
The sequences of primers used can be found in
4
Quantitative RT-PCR Analysis of Gene Expression
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RNA was extracted with the RNeasy Plus mini kit (Qiagen). cDNA was synthesized by random priming from 1 µg of total RNA with the Q-Script cDNA super mix kit (Quanto biology), according to the manufacturer’s protocols. Primers for qPCR analysis were synthesized by the Eurofins Scientific. We performed qPCR using PerfeCTa SYBR Green FastMix (Bio-Rad), according to the manufacturer’s protocol. We analyzed data, determining quantities of gene-specific mRNA expression, using the comparative CT method as described previously35 (link). CT refers to the “threshold cycle” and is determined for each experiment with MyiQ software. Amplification of GAPDH was performed for each reverse-transcribed sample as an endogenous quantification standard. The primers are as follows: GFP, 5′-TCAAGAGTGCCATGCCTGAA-3′ and 5′-TGGTCTGCTAGTTGAACGCT-3′, Syn1 (both endogenous Syn1 and Syn1f), 5′-ATGGAGCCCAAGATGCA-3′ and 5′-AGATCGTGGGCTAGCAG-3′, ASCT2, 5′-CACCATGGTTCTGGTCTCCT-3′ and 5′-GCGGGTGAAGAGGAAGTAG-3′ and GAPDH, 5′-ATTGACCTCAACTACATGGTTTACATG-3′ and 5′-TTGGAGGGATCTCGCTCCTGGAAG-3′.
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