Both cultured control and siGLUL transfected cells were detached from plates after 48 h with 0.25% EDTA-trypsin (DENAzist-Asia, Mashhad, Iran), and the total RNA was isolated using column RNA extraction kit (DENAzist-Asia, Mashhad, Iran) according to the company's instructions. The cDNA was subsequently synthesized with 1 μg of extracted RNA and by a cDNA easy synthesis kit for further specific studies (Parstoos, Mashhad, Iran). The quantitative RT-PCR was administered with primers for target genes, including GLUL, GSTMu3, ENO1 and Bax, and β-actin using High ROX RealQ Plus 2x Master Mix Green (AMPLIQON; Denmark) by ABI step one plus (Applied Biosystems, USA). Primer sequences and the conditions of qRT-PCR are available in Table 1(Tab. 1) (References in Table 1: Huang et al., 2019[29 (link)]; Kung et al., 2011[35 (link)]; Wang et al., 2020[70 (link)]). Studied genes were normalized based on the housekeeping gene β-actin as the reference gene. The relative expression values of mRNA were determined using the 2−∆∆Ct method between the siGLUL transfected and control cells.
Realq plus 2x master mix green high rox
Manufactured by Ampliqon
Sourced in Denmark, United States
RealQ Plus 2x Master Mix Green High ROX is a ready-to-use reaction mixture for quantitative real-time PCR (qPCR) applications. It contains a DNA polymerase, dNTPs, and a high ROX reference dye, all pre-optimized for reliable and sensitive qPCR results.
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus instrument, by Thermo Fisher Scientific (3 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (2 mentions)
Abi steponeplus, by Thermo Fisher Scientific (1 mentions)
Abi 7500 real time analyzer, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rotor gene q, by Qiagen (1 mentions)
3730xl sequencer, by Thermo Fisher Scientific (1 mentions)
Propidium iodide solution, by Merck Group (1 mentions)
Gapdh 2118, by Cell Signaling Technology (1 mentions)
Trypsin edta, by Biosera (1 mentions)
Caspase 3 9662, by Cell Signaling Technology (1 mentions)
Fbs fetal bovine serum, by Thermo Fisher Scientific (1 mentions)
Vincristine vcr, by Merck Group (1 mentions)
Penicillin streptomycin, by Biosera (1 mentions)
Annexin 5 apoptosis detection kit, by BD (1 mentions)
3730xl sequencer, by Macrogen (1 mentions)
Endofree plasmid mega kit, by Qiagen (1 mentions)
1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride, by Merck Group (1 mentions)
Succinic anhydride, by Merck Group (1 mentions)
10 protocols using realq plus 2x master mix green high rox
1
Quantitative RT-PCR Analysis of Gene Expression
2
Quantifying MLL5 and E6 Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Real time PCR was our method of choice to evaluate the expression of MLL5 and E6 genes in treated target cells. Seventy-two hours after transfection, HeLa cells were subjected to total RNA extraction procedure, using TRYzol RNA Extraction Reagent (Dara Zistfan Eram; Shiraz, Iran). Complementary DNA (cDNA) was synthesized, using PrimeScript 1st strand cDNA Synthesis Kit (Takara, Japan), according to the manufacturer's instructions. qPCR was performed on ABI 7500 Real Time Analyzer (Applied Biosystem, USA), using high-ROX RealQ Plus 2x Master Mix Green (Ampliqon, Denmark) and specific primers for MLL5, E6, and Beta-2-microglobulin (B2M) genes (Table 1(Tab. 1) ). The experiments were performed independently in a quadruplicate manner.
3
Evaluating HLA-A Knockout Impact
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the impact of gene knockout on HLA-A gene expression, real-time PCR was carried out in treated target cells. HEK293T cells were harvested by trypsin, and RNA was extracted using RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized, using Prime-Script 1st strand cDNA Synthesis Kit (Takara, Japan), according to the manufacturer's instructions. Real-time PCR was performed on QuantStudio 3 Real-Time PCR System (Applied Biosystem, USA), using high-ROX RealQ Plus 2x Master Mix Green (Ampliqon, Denmark) and specific primers for HLA-A and Beta-2microglobulin (B2M) genes (forward: 5′-ACCGTC CAGAGGATGTATG-′3, reverse: 5-′CTTTCAGGGC-GATGTAATC-′3). The experiments were performed independently in a duplicate manner, and the relative expression of genes was analyzed using the 2-ΔΔct method [13] (link).
4
Gene Expression Analysis by qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genes expression levels was measured by Real-Time PCR (qRT-PCR) (Rotor Gene Q, Qiagen, Germany). Real Q Plus 2x Master Mix Green-high Rox™ (Ampliqon, Denmark), cDNA, and synthesized primers were used for this step. The temperature profile was as follows: initial denaturation at 95 °C for 15 min followed by 40 consecutive cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, and extension at 72 °C for 20 s. The amplification curve of each PCR reaction was normalized with the amplification curve of the GAPDH reference gene. The 2−∆∆CT formula was also used to determine the gene expression in the present study.
5
Quantifying lncRNA Expression by qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The relative expression of lncRNA UCA1 and ecCEBPA were measured by quantitative real-time RT-PCR with specific primers designed using the GeneRunner software package, version 4.0 (Table 1 ). Primers for amplification of the GUSB (β-Glucuronidase) gene (as an internal control) were taken from another study (24 (link)). PCR was performed using RealQ Plus 2x Master Mix Green (high Rox) (Ampliqon, Odense M, Denmark) on an Applied Biosystems StepOnePlus™ instrument. The PCR cycling conditions consisted of a first denaturation step at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 sec, annealing at 61°C for lncRNA UCA1 and ecCEBPA, and at 60 °C for GUSB genes and then extension for 15 sec at 72 °C. Additionally, the specificity of PCR amplicons was verified by Sanger sequencing using Applied Biosystems 3730XL sequencer (Macrogen, Seoul, South Korea).
6
Anticancer Effects of Vincristine in vitro
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell culture media, penicillin-streptomycin, and trypsin EDTA were purchased from Biosera (Ringmer, UK). Bovine Fetal Serum (FBS) was purchased from Gibco, Thermo Fisher Scientific (Germany). The MTT powder_2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide_ was purchased from MELFORD (United Kingdom). Vincristine (VCR) and Propidium iodide solution were purchased from Sigma Aldrich (Germany). Also, Caspase-3: 9662S, NF-κBp56: 3033T, and GAPDH: 2118S antibodies applied in western blot analysis were purchased from Cell Signaling Technology company (USA). ROS assay Kit was purchased from Teb Pazhouhan Razi Company. Annexin V apoptosis detection kit was purchased from the BD Biosciences company. RealQ plus 2x master mix green high ROX and RevertAid Reverse transcriptase (RT) were purchased from ampliqon (Denmark) and Thermo Fisher Scientific (USA), respectively. TRIzol Reagent was purchased from Zaver Zist Azma(Iran).
7
Quantitative Analysis of Circular RNAs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Applying the relative quantitative real-time RT-PCR, the expression levels of cir_ITCH and circHIPK3 were assessed compared to GUSB (β-Glucuronidase) as an internal control (23). Divergent primers, rather than convergent primers, were designed with GeneRunner software, version 4.0 to amplify the circular RNAs. Basic local alignment search tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used for confirmation of unity attachment of divergent primers to genome. A set of convergent primers in an opposite direction were designed to amplify only the linear forms. The sequences of primers are listed in Table 1. RealQ Plus 2x Master Mix, green (high ROX) (AMPLIQON, Odense M, Denmark) was applied in an Applied Biosystems StepOnePlus™ instrument for PCR amplification. The amplification conditions consisted of an initial denaturation at 95 °C for 2 min, then 30 cycles of denaturation at 95 °C for 30 s, annealing at 55.9 °C for cir_ITCH and circHIPK3 and at 60 °C for GUSB genes for 60 s, and the extension step for 30 s at 72 °C. Furthermore, the PCR products of some samples were sequenced with an Applied Biosystems 3730XL sequencer (Macrogen, Seoul, South Korea) to verify the specific amplification of circular RNAs.
8
Branched PEI-Mediated CD200 Gene Delivery
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Branched PEI (LMW PEI; average MW = 1,800 Da), succinic anhydride, MTT, N-hydroxybenzotriazole, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride, 1-hydroxybenzotriazole hydrate, and N-[2-hydroxythyl] piperazine-N-[2-ethanesulfonic acid] (HEPES) were purchased from Sigma-Aldrich (Munich, Germany). Human CD200 plasmid (pCMV-XL5-hCD200) was obtained from OriGene (Rockville, MD, USA). Cell culture experiments were performed using FBS and DMEM (Gibco, Gaithersburg, MD, USA). Spectra/Por dialysis membrane was obtained from Spectrum Laboratories (Houston, TX, USA). Transfor-mAid Bacterial Transformation Kit (K-2710) was purchased from Thermo Scientific Company (Hanover, MD, USA). Plasmid purification was performed by Qiagen Endofree Mega Plasmid Kit (Qiagen, Hilden, Germany). cDNA synthesis and Real Time PCR were performed using the PrimeScript™ RT reagent Kit (Perfect Real Time, TaKaRa, Dalian, People’s Republic of China), and RealQ Plus 2x Master Mix Green High ROX™ (AmpliQon, Denmark). All solvents and chemicals were purchased from Sigma-Aldrich (Munich, Germany) and were of the highest purity available.
9
Gene Expression Analysis via qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primers were designed using DNASTAR Lasergene software and synthesized by Metabion (Germany). Real-time reactions were made using RealQ plus 2X Master Mix Green high ROX (Ampliqon, Denmark) according to the manufacturer's instruction. For fluorescent reporter dye, the real-time master mix had SYBR Green. Collected relative gene expression data were analyzed by the 2 -ΔΔCT (Livak) method, and statistical analyses were performed using GraphPad Prism 8.
10
Quantitative RT-PCR Analysis of Long Non-Coding RNAs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tissues were cut into 30-35 mg polymerase chain reaction pieces and homogenized using an automated homogenizer (Precelly®24; Bertin Technologies, France) with QIAzol® lysis reagent (Qiagen, USA). The RNA extraction was then performed according to the manufacturer's instructions.
Complementary DNA (cDNA) synthesis was performed on DNase-treated RNA by using random hexamer primers and an M-MLV reverse transcriptase (Yekta Tajhiz Azma, Iran). Quantitative real time RT-PCR was performed using RealQ Plus 2x Master Mix Green High ROX (Ampliqon, Denmark) and speci c primers for candidate lncRNAs and hypoxanthine guanine phosphoribosyl transferase (Hprt) as a reference gene [37] (link) (Table 1) on an Applied Biosystems StepOnePlus™ instrument. The PCR ampli cation conditions consisted of an initial denaturation at 95°C for 15 minutes, 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. Furthermore, to make sure that the actual genes of interest were speci cally ampli ed, the PCR products of some specimens were sequenced with an Applied Biosystems 3500 sequencer (Pishgam Biotech Co., Tehran, Iran). The BioEdit sequence alignment editor [38] and nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) were applied to analyze the results.
Complementary DNA (cDNA) synthesis was performed on DNase-treated RNA by using random hexamer primers and an M-MLV reverse transcriptase (Yekta Tajhiz Azma, Iran). Quantitative real time RT-PCR was performed using RealQ Plus 2x Master Mix Green High ROX (Ampliqon, Denmark) and speci c primers for candidate lncRNAs and hypoxanthine guanine phosphoribosyl transferase (Hprt) as a reference gene [37] (link) (Table 1) on an Applied Biosystems StepOnePlus™ instrument. The PCR ampli cation conditions consisted of an initial denaturation at 95°C for 15 minutes, 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. Furthermore, to make sure that the actual genes of interest were speci cally ampli ed, the PCR products of some specimens were sequenced with an Applied Biosystems 3500 sequencer (Pishgam Biotech Co., Tehran, Iran). The BioEdit sequence alignment editor [38] and nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) were applied to analyze the results.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!