Total RNA was isolated using a Favorgen RNA extraction kit (#FKBRK 001, Favorgen Biotech) used in accordance with the manufacturer's instructions from the harmaline‐treated A2780 cells (150 and 300 μM). The purity and concentration of the extracted RNA were assessed using UV spectrophotometry (NanoDrop 1000TM, USA) and agarose gel electrophoresis. An easy cDNA synthesis kit (#A101161, Parstous, Iran) was used to reverse transcribe total RNA into cDNA. RealQ Plus 2X‐MasterMix Green without RoxTM (#A323402, Amplicon, Denmark) was then used for qRT‐PCR (Roche, USA), with specific primers for the genes GAPDH, Bax, Bcl‐2, P53, MMP‐2, and MMP‐9, listed in Table 1 (Metabion, Germany). The Livak technique was used to evaluate the relative expression of genes.
Favorgen rna extraction kit
Manufactured by Favorgen Biotech
Sourced in Singapore
The Favorgen RNA extraction kit is a laboratory equipment designed for the efficient extraction and purification of RNA from various biological samples. The kit utilizes a specialized protocol and reagents to isolate high-quality RNA for downstream applications such as gene expression analysis, reverse transcription, and other molecular biology experiments.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrometer, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Abi prism 7900, by Thermo Fisher Scientific (1 mentions)
Real time pcr system software, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Uv spectrophotometry, by Thermo Fisher Scientific (1 mentions)
Allstars negative sirna af 488, by Qiagen (1 mentions)
6 protocols using favorgen rna extraction kit
1
Analyzing Gene Expression in Harmaline-Treated A2780 Cells
2
Bactericidal Ability of Macrophages
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To determine the bactericidal ability of macrophages, 2 × 105 gram-negative (E. coli) or 2 × 105 gram-positive (L. monocytogenes) bacteria were incubated with BMDMs for 2 hours. Cells were then thoroughly washed with PBS for 3 to 5 times and incubated for 16 to 24 hours in DMEM containing antibiotics before harvesting for intracellular bacteria. Subsequently, cell lysate from macrophages containing intracellular bacteria was serially diluted with PBS and spread onto agar plates to determine bacterial viability (expressed as CFU). For qRT-PCR analysis, total RNA was prepared using Favorgen RNA extraction kit (Favorgen Biotech, catalog no. FATRK 001–2).
3
Quantitative Gene Expression Analysis of Heart and Macrophage
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Total RNA from heart tissues (left ventricles) and BMDMs were isolated using TRIzol (Life Technologies, Singapore, catalog no. 15596018) and Favorgen RNA extraction kit (Favorgen Biotech, catalog no. FATRK 001–2), respectively. The concentration and quality of RNA were measured with UV spectrophotometry (NanoDrop Technologies, Wilmington, North Carolina, USA). For cDNA synthesis, total RNA was reverse transcribed using random hexamers and SuperScript III FirstStrand Synthesis system (Life Technologies, catalog no. 18080–051). Gene expression was performed by quantitative RT-PCR (ABI PRISM 7900 or ViiA7 Real-Time PCR System) using 10-μl reaction mixture containing 20 ng cDNA, SYBR Green Master Mix (Life Technologies, catalog no. 4368702), 6-μM forward primer, and 6-μM reverse primer. Results were analyzed with Real-Time PCR System Software (Applied Biosystems, Singapore). All mRNA data were normalized against the reference gene Gapdh.
4
RNA Extraction and cDNA Synthesis from Promastigotes
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RNA was extracted from samples containing 1 × 106 Stationary-phase promastigotes using a Favorgen RNA extraction kit (Favorgen Biotech) according to the manufacturer’s instructions. Re-suspension was done in 60 µl of nuclease-free water. RNA quantity was checked by the NanoDrop spectrometer (Thermo Scientific Fisher, USA), and based on the results, 1–2 µL was used for cDNA synthesis. The cDNA was then synthesized with a combination of oligo (dT) 18 and random hexamer primers using the cDNA Synthesis Kit (YTA, Iran, Cat No: YT4500) according to the manufacturer’s recommendation.
5
Total RNA Extraction and cDNA Synthesis
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Total RNA from 1-5 Â 10 6 promastigotes in the stationary phase was isolated according to the manufacturer's instructions (Favorgen RNA extraction kit, Favorgen Biotech). Isolated RNA was resuspended in 60 mL of nuclease-free water. The quantity of the extracted RNAs was verified by a NanoDrop spectrometer (Thermo Scientific Fisher, USA). Based on the quantity of the extracted RNA, 1 to 2 mL were used for cDNA synthesis. The cDNA was then synthesized with a combination of oligo (dT) 18 and random hexamer primers using the cDNA Synthesis Kit (YTA, Iran with Cat No: YT4500) according to the manufacturer's instructions.
6
Modulation of Macrophage Polarization
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Control (AllStars Negative siRNA AF488, catalog no. 1027284), YAP siRNAs (catalog no. GS22601), and TAZ siRNA (catalog no. SI01442511 and SI01442532) were obtained from Qiagen, USA. HDAC3 silencer pre-designed siRNA (catalog no. AM16708) and silencer negative control siRNA (catalog no. AM4620) were purchased from Invitrogen, USA. The siRNA was transfected in BMDMs using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher, Singapore, catalog no. 13778150). Briefly, after a transient transfection, BMDMs were incubated for 72 hours and treated with either pro-inflammatory (LPS 100 ng/ml + IFNγ 10 ng/ml) or reparative stimuli (IL4 10 ng/ml + IL13 10 ng/ml) diluted in culture medium (DMEM containing 10% FBS and 1% penicillin/streptomycin). Cells were collected for total RNA isolation and qRT-PCR analysis using Favorgen RNA extraction kit (Favorgen Biotech, Taiwan, catalog no. FATRK 001–2). Cell lysates were prepared using RIPA buffer (Thermo Fisher, catalog no. 89901) for western blot analysis.
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