Mycycler pcr system

Manufactured by Bio-Rad

Sourced in United States, Japan

The MyCycler PCR system is a thermal cycler device designed for amplifying DNA sequences using the polymerase chain reaction (PCR) technique. It provides the essential functions required for performing PCR experiments, including precise temperature control and cycling capabilities.

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MyCycler (172 citations) Bio-Rad MyCycler PCR Thermal Cycler system (4 citations) MyCycler PCR (2 citations) MyCycler system (2 citations) MyCycler real-time PCR system (2 citations)

7 protocols using mycycler pcr system

Isolation and Quantification of Circulating miRNA

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Total RNA was isolated using RNAiso Plus (Takara, Japan). Circulating microRNA in peripheral blood was isolated using the mirVana PARIS Kit (Ambion, USA). Reverse transcription reactions were performed using the PrimeScript™ RT reagent kit with gDNA Eraser (Takara) for mRNA and Bulge-Loop™ miRNA qRT-PCR Starter kit (RiboBio) for miRNA to make cDNA from total RNA in a MyCycler PCR system (Bio-Rad, USA). Subsequently, quantitative real-time PCR was performed using SYBR^® Premix Ex Taq II (Takara). Primer pairs for miR-181b-5p, cel-miR-39 and U6 were purchased from RiboBio Co. Ltd. Primer pairs for GAPDH and genes associated with stemness and EMT were designed by Sangon Biotech Co. Ltd. (China). Each sample was performed in triplicate, and the reaction products were analyzed using the ABI 7500 Prism Sequence Detection system (Applied Biosystems, USA). Data analysis was based on the Ct method (∆∆Ct according to Applied Biosystems). All operations followed the manufacturer's protocol.

Li X., Han J., Zhu H., Peng L, & Chen Z. (2017). miR-181b-5p mediates TGF-β1-induced epithelial-to-mesenchymal transition in non-small cell lung cancer stem-like cells derived from lung adenocarcinoma A549 cells. International Journal of Oncology, 51(1), 158-168.

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Evaluating mtDNA Damage via Long PCR

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Long PCR was performed using an ApexHF HS DNA Polymerase CL Kit (Accurate Biotechnology, China) in a MyCycler PCR system (Bio-Rad, USA) for mtDNA damage evaluation, according to the protocol described previously (Zhou et al., 2011 (link)). The primers are listed in Table 1. The final long PCR cycling parameters followed the manufacturer’s recommendations: initial denaturation of 1 min at 94°C followed by 10 s at 98°C, 15 s at 60°C, and 12 min at 68°C for 28 cycles. The PCR cycling parameters for small fragments underwent the following profiles: initial denaturation of 1 min at 94°C followed by 10 s at 98°C, 15 s at 60°C, and 30 s at 68°C for 25 cycles. Each PCR product was quantified using ImageJ.

Ren Y, & Zhu S. (2022). Nitric oxide promotes energy metabolism and protects mitochondrial DNA in peaches during cold storage. Frontiers in Plant Science, 13, 970303.

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Quantitative RT-PCR Analysis of eEF2K in HepG2 Cells

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Total RNA samples from HepG2 and HepG2/CDDP cells were extracted using RNAiso Plus reagent following the manufacturer’s protocols. RNA (1 μg) was reverse-transcribed using a ReverTra Ace qPCR RT-Kit (Toyobo Life Science, Osaka, Japan) in a MyCycler PCR system (BioRad Laboratories, Hercules, CA). SYBR Green PCR Master Mix was purchased from Toyobo Life Science. The 2^−ΔΔCT cycle threshold method was used for the calculation of relative differences in mRNA abundance with a LightCycler 480 qPCR System (Roche Molecular Biochemicals, Mannheim, Germany). Data were normalized to the expression of β-Actin. The results of qRT-PCR were expressed as fold-changes. The normalized value of the target mRNA of the control group is arbitrarily presented as 100. The pairs of primer for PCR were listed below:

eEF2K: (sense) 5′-GGCAAACTCCTTCCACTTCA-3′;

eEF2K: (antisense) 5′-CATCATCCAGCCATTCCC-3′.

β-Actin: (sense) 5′-GCACCACACCTTCTA CAATG-3′;

β-Actin: (antisense) 5′-TGCTTGCTGATCCACATCTG-3′.

Zhang C., Lei J.L., Zhang H., Xia Y.Z., Yu P., Yang L, & Kong L.Y. (2017). Calyxin Y sensitizes cisplatin-sensitive and resistant hepatocellular carcinoma cells to cisplatin through apoptotic and autophagic cell death via SCF βTrCP-mediated eEF2K degradation. Oncotarget, 8(41), 70595-70616.

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Total RNA Extraction and RT-PCR

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Total RNA was prepared with TRIzol reagent following the manufacturer’s protocols. RT-PCR was carried out with MyCycler PCR System (Bio-Rad, Hercules, CA, USA) as previously described [38 (link)]. The primer sequences used are listed in Supplementary Table S1.

Wu J., Du J., Li Z., He W., Wang M., Jin M., Yang L, & Liu H. (2022). Pentamethylquercetin Regulates Lipid Metabolism by Modulating Skeletal Muscle-Adipose Tissue Crosstalk in Obese Mice. Pharmaceutics, 14(6), 1159.

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Characterization of Cannabinoid Receptor Plasmid

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Standard laboratory chemicals and reagents were obtained pure or at the highest available grade from commercial sources.^{24 (link)–28 (link),31 (link)} Gen Elute plasmid mini-prep kit, Dulbecco’s modified Eagle’s medium (DMEM), and cAMP kit were from Sigma (St. Louis, MO). AM3677 (Figure 1) was synthesized at the Center for Drug Discovery, Northeastern University (Boston, MA). CP55,940 and [³H]CP55,940 were obtained from the National Institute on Drug Abuse (Bethesda, MD). pRC/CMV-hCB1R was a generous gift from Dr. T.I. Bonner (National Institute of Mental Health, Bethesda, MD). Oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA). The My Cycler PCR system was purchased from BioRad Laboratories (Hercules, CA). Pfu Turbo DNA polymerase was purchased as part of the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). One Shot Top10 Escherichia coli cells, Flp-In-293 cells, pcDNA/FRT, hygromycin, lipofectamine 2000, and pure link hipure plasmid filter midi-prep kit were purchased from Invitrogen (Carlsbad, CA). MinElute gel-extraction kits and the QIA Prep spin mini-prep kits were from Qiagen (Valencia, CA). Restriction endonucleases were purchased from New England Biolabs (Beverly, MA). Fetal bovine serum (FBS) and penicillin-streptomycin solution were purchased from GIBCO (Rockville, MD).

Janero D.R., Yaddanapudi S., Zvonok N., Subramanian K.V., Shukla V.G., Stahl E., Zhou L., Hurst D., Wager-Miller J., Bohn L.M., Reggio P.H., Mackie K, & Makriyannis A. (2015). Molecular-Interaction and Signaling Profiles of AM3677, a Novel Covalent Agonist Selective for the Cannabinoid 1 Receptor. ACS chemical neuroscience, 6(8), 1400-1410.

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Quantifying metabolic gene expression

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Total RNA samples from U2OS, SW1353 and S180 cells were extracted using RNAiso Plus reagent following the manufacturer's protocols. RNA (1 μg) was reverse-transcribed using a ReverTra Ace qPCR RT-Kit (Toyobo Life Science, Osaka, Japan) in a MyCycler PCR system (BioRad Laboratories, Hercules, CA). SYBR Green PCR Master Mix was purchased from Toyobo Life Science. The 2^−ΔΔCT cycle threshold method was used for the calculation of relative differences in mRNA abundance with a LightCycler 480 qPCR System (Roche Molecular Biochemicals, Mannheim, Germany). Data were normalized to the expression of β-actin. The results of real-time PCR were expressed as fold-changes. The normalized value of the target mRNA of the control group is arbitrarily presented as 1. The pairs of primer for PCR were listed below:

c-myc: (sense) 5′-TGGTGCTCCATGAGGAG ACA-3′;

c-myc: (antisense) 5′-GTGTTTCAACTGTTCT CGTC-3′.

β-actin: (sense) 5′-GCACCACACCTTCTA CAATG-3′;

β-actin: (antisense) 5′-TGCTTGCTGATCCACATC TG-3′.

HK II: (sense) 5′-ACAATGGATGCCTAGATG-3′

HK II: (antisense) 5′-AGGTACATTCCACTG ATC-3′

PFKP: (sense) 5′-ACCACCGATGATTCCATT-3′

PFKP: (antisense) 5′-CTTGAGCCACCACTGT TC-3′

LDHA: (sense) 5′-TGGTTGAGAGTGCTTATG-3′

LDHA: (antisense) 5′-GCCTAAGATTCTTCAT TATACT-3′

PKM2: (sense) 5′-CCACTTGCAATTATTTGA GGAA-3′

PKM2: (antisense) 5′-GTGAGCAGACCTGCCAGACT-3′

Zhang C., Yang L., Geng Y.D., An F.L., Xia Y.Z., Guo C., Luo J.G., Zhang L.Y., Guo Q.L, & Kong L.Y. (2016). Icariside II, a natural mTOR inhibitor, disrupts aberrant energy homeostasis via suppressing mTORC1-4E-BP1 axis in sarcoma cells. Oncotarget, 7(19), 27819-27837.

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Quantification of PD-L1 and IFN-γ mRNA

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To quantify PD-L1 mRNA expression, total RNA was isolated and cDNA was synthesized using TaqMan MultiScribe Reverse Transcriptase (Applied Biosystems, FosterCity, CA) as previously described [51 (link)]. Quantitative real-time PCR analysis was performed using an ABI Prism 7900-HT Sequence Detection System (96-well, AppliedBiosystems) and Semi-quantitative PCR was performed using Bio-Rad MyCycler PCR System. Primers for this study included: forward primer 5#-CCTACTGGCATTTGCTGAACGCAT-3# and reverse primer 5#-ACCATAGCTGATCATGCAGCGGTA -3# for PD-L1; forward primer 5#-CTCTTGGCTGTTACTGCCAGG-3# and reverse primer 5#-CTCCACACTCTTTTGGATGCT-3# for IFN-γ. Primers used for β-actin were previously reported [51 (link)]. The total semi-quantitative PCR product was then run on a 2% agarose gel.

Fang W., Zhang J., Hong S., Zhan J., Chen N., Qin T., Tang Y., Zhang Y., Kang S., Zhou T., Wu X., Liang W., Hu Z., Ma Y., Zhao Y., Tian Y., Yang Y., Xue C., Yan Y., Hou X., Huang P., Huang Y., Zhao H, & Zhang L. (2014). EBV-driven LMP1 and IFN-γ up-regulate PD-L1 in nasopharyngeal carcinoma: Implications for oncotargeted therapy. Oncotarget, 5(23), 12189-12202.

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