In order to evaluate miRNA-223 expression, we extracted total RNA from 250 μL of the serum using the RNX-Plus (SINACLON, RN7713C/EX6101, Karaj, Iran) kit according to the manufacturer’s protocol with some modifications such as increasing the time of incubation and centrifugation to obtain the highest amount of miRNAs in the samples. The quantity and quality of the extracted RNAs was assessed using NanoDrop 2000 (Thermo, Wilmington, DE, USA), and samples with absorbance of 1.8–2 at 260/280 nm were used for miRNA specific cDNA synthesis.
Rnx plus
Manufactured by Sinaclon
Sourced in United States
The RNX-Plus is a high-precision laboratory equipment designed for scientific research and analysis. It is a multi-functional device capable of performing various tasks related to the study and manipulation of ribonucleic acid (RNA). The core function of the RNX-Plus is to facilitate the extraction, purification, and quantification of RNA samples. This equipment is widely used in fields such as molecular biology, genetics, and biotechnology.
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Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Rotor gene 3000, by Bio-Rad (2 mentions)
Cdna synthesis kit, by Sinaclon (2 mentions)
Rotor gene 6000, by Qiagen (2 mentions)
Random hexamer primer, by Thermo Fisher Scientific (2 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Iq5 optical system software version 2, by Bio-Rad (2 mentions)
Rq1 dnase 1, by Sinaclon (2 mentions)
Realq plus 2x master mix green, by Ampliqon (2 mentions)
Accupower rt premix, by Bioneer (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Hela cell, by American Type Culture Collection (1 mentions)
Lipofectamine transfection reagent, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Mouse anti his tag antibody, by Bio-Rad (1 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Abi prism 7500 rt pcr system, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
24 protocols using rnx plus
1
Extracting miRNA-223 from Serum
2
Chondrogenic Potential of IPFP-ASCs Scaffold
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After 21 days, each IPFP-ASCs seeded scaffold was homogenized under liquid nitrogen using a mortar. RNA was isolated from the samples by using RNX-Plus (Sinaclon, IRAN) according to the manufacturer’s instructions. The concentration of RNA was estimated spectrophotometrically with NanoDrop 1000 Spectrophotometer (Wilmington, DE, USA) at A260/280. The RNA samples were reverse transcribed into first-strand cDNA using the AccuPower® RT PreMix (Bioneer). Real Time-PCR reactions were performed using the SYBRGreen PCR Mastermix (Applied Biosystems, USA), according to the manufacturer’s instructions. These gene primers were purchased from (Bioneer). The cartilage-specific oligonucleotide primers which were used, were aggrecan (forward 5’-AGGGCGAGTGGAAT-GATGTT-3’; reverse 5’-GGTGGCTGTGCCCTTTTTAC-3’), and collagen type 2a1 (forward 5’-ATGCCACA-CTCAAGTCCCTCAA-3’; reverse 5’-CGCAAGTCTCG-CCAGTCTCC-3’), (IHH) (forward 5’-CACTTCTGCC-TGGTCCTGTT-3’; reverse 5’-GGGCTGAACTGCTTG-TAGG-3’), housekeeping gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward, 5’-CAAGATCATCAGCAATGCCTCC-3’; reverse, 5’-GC-CA-TCACGCCACAGTTTCC-3’). All experiments were performed in triplicate for each sample. Interpreta-tion of the results was performed using the Pfaffle method and the CT values were normalized with respect to GAPDH expression.
3
In Vitro Expression of Mtb32C-HBHA Fusion Genes
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To confirm the in vitro expression of Mtb32C-HBHA fusion genes, the plasmid was transfected into HeLa cells (American Type Culture Collection Manassas, VA, USA) using lipofectamine transfection reagent according to the manufacturer’s instructions (Invitrogen, USA). Then, 72 hr after transfection, the cells were treated with 0.5 ml trypsin (Invitrogen, USA) and were incubated for 10 min. Cell suspension was harvested and total RNA was extracted using RNX-Plus (SinaClon, Iran), as described previously (16 (link), 17 (link)). Purified RNA was used for cDNA synthesis using cDNA synthesis kit (Pars Tous, Iran) and was amplified by PCR. To detect the presence of His-Tag marker in chimeric Mtb32C-HBHA protein (a marker of constructed rather than natural protein), Western blot method was performed using mouse anti-His Tag antibody as the primary antibody and peroxidase conjugated rabbit anti-mouse IgG as the secondary antibody (AbD SeroTec, USA).
4
Placental Total RNA Isolation and qRT-PCR
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To isolate total RNA from the placenta, we used RNX-Plus (Sinaclon, Tehran, Iran). Moreover, PrimeScript 1st strand cDNA synthesis kit (Takara Bio, Shiga, Japan) was used to generate cDNA according to the manufacturer’s instructions. An ABI PRISM 7500 RT-PCR system (Applied Biosystems) with SYBR Green was used to evaluate mRNA expression. For amplification, a reaction mixture (20 μl) of SYBR Green/high ROX (10 μl; Amplicon), 2 μl of cDNA solution, 7 μl of nuclease-free water, and 10 pmol of each primer [23 (link)–25 (link)] was used. Analysis of each sample was performed in triplicate. To determine the relative expression of mRNA in the target genes, the 2-ΔΔCt method was applied, then, the expression levels were normalized to a housekeeping gene; ΔCt was the difference between β-actin (control) and P21/TP53 genes.
5
SKBR3 and CHO Cell Transfection
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The SKBR3 and CHO cell lines were obtained from the National Cell Bank of Iran (Pasteur Institute of Iran, Tehran, Iran), DTX from AQVida® (Hamburg, Germany), and human IGF-1Rα/β siRNA (sc-29358) and scrambled siRNA (sc-37007) were obtained from Santa Cruz Biotechnology® (Santa Cruz, CA, USA). The MUC1-specific DNA Apt (5’-Amino-C6-GGG AGA CAA GAA TAA ACG CTC AAG AAG TGA AAA TGA CAG AAC ACA ACA TTC GAC AGG AGG CTC ACA ACA GGC-3’) was purchased from TAG A/S® (Copenhagen, Denmark). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), membrane dialysis bag (12 kDa cut-off), and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were procured from Merck® (Hohenbrunn, Germany). RPMI-1640, Trypsin-EDTA (0.25%), and fetal bovine serum (FBS) were obtained from Gibco® (Gibco, Canada), green fluorescence protein (GFP)-containing plasmid (pEGFP-N1 vector) from Clontech Laboratories® (CA, USA), and RNX-Plus from Sinaclon (Tehran, Iran). All other reagents were of analytical grade.
6
Hepatic Gene Expression Quantification
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Real-time PCR calculated hepatic TNF-alpha, IL-1, α-SMA, and TGF-β mRNA. Total RNA was isolated from hepatic tissues using RNX Plus (Sinaclon, Tehran, Iran) and cDNA was generated on the basis of the manufacturer's technique from total RNA by cDNA Synthesis kit (Sinaclon, Tehran, Iran). RT-PCR was conducted using the instrument Rotor Gene 3000 (Bio-Rad, USA). The mRNA values of TNF-α, IL-1, α-SMA, and TGF-β were standardized to GAPDH. The PCR was completed in 40 cycles: TNF-α and IL-1 at 95°C for 15 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, α-SMA at 95°C for 15 seconds, 63°C for 30 seconds, and 63°C for 30 seconds, and TGF-β at 95°C for 15 seconds, 55°C for 30 seconds, 72°C for 30 seconds. Data of TNF-α, IL-1, α-SMA, and TGF-β were calculated relative to GAPDH using the 2–ΔCt method.
7
Testis RNA Extraction and cDNA Synthesis
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RNA extraction and cDNA synthesis. Total RNA was extracted from the mice testis using RNX plus (EX6101) solution following the manufacturer’s protocol (SINACLON, Iran). The RNA pellet was dissolved in RNase-free water (DEPC treated water). Total RNA was then treated with DNAaseI (SINACLON, Iran) to destroy possible genomic DNA contamination followed by heat treatment (65°C for 10 minutes) to inactivate the enzyme and then stored at −80°C for future cDNA synthesis. We checked the purity and integrity of the total RNA extracted at 260/280 nm ratio using a Thermo Scientific NanoDrop spectrophotometer and visualized it on 1% agarose gel. We used a commercial cDNA synthesis kit (2-steps RT-PCR kit, Vivantis, Malaysia) following the manufacturer’s instructions. Briefly, 1 µg of total RNA, 0.5 µl M-MuLV Reverse Transcriptase (200 U/µl), 2 µl of 10X Buffer M-MuLV, 1 µl Oligo d(T)18 (40 µM), 1 µl of 10 mM dNTPs were added to a 0.2 ml microcentrifuge tube. The reaction mixture was adjusted to the final 20 µl volume with nuclease-free water.
8
Hepatic Gene Expression Analysis
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Total RNA was separated from hepatic tissues utilizing RNX Plus (Sinaclon, Tehran, Iran) and cDNA was produced from total RNA by cDNA Synthesis kit (Sinaclon, Tehran, Iran) based on the manufacturer's procedure. RT-PCR was done by Rotor Gene 3000 instrument (Bio-Rad, USA), and data of TNF-α, α–SMA, IL-1, and TGF-β were determined in comparison to GAPDH using the 2–ΔCt procedure.
9
Quantitative Gene Expression Analysis
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RNX-Plus (SinaClon; RN7713C) Kit was used to extract total RNA from homogenized liver tissue. The quantity and quality of extracted RNAs was determined using a nanodrop ND-1000 spectrophotometer (Thermo science, Newington, NH). Electrophoresis on 1% agarose gel was also performed to determine the quality of extracted RNAs. Revert aid reverse transcriptase (Thermo science, Germ any) and random hexamer primers (Thermo science, Germ any) were used for cDNA synthesis at 42 °C for 1 h. A Rotor Gene 6000 (Corbett Research, Australia) thermocycler in 40 cycles was applied for amplifications. Each reaction included 5 μL master mix and 100 nM primers. Primer sequences are as follow [20 (link)–22 (link)]: Nrf2, 5/-CACATCCAGACAGACACCAGT-3/ (forward), 5/-CTACAAATGGGAATGTCTCTGC-3/ (reverse); NLRP3, 5/-GAGCTGGACCTCAGTGACAATGC-3/ (forward), 5/- ACCAATGCGAGATCCTGACAACAC-3/ (reverse); Caspase 1, 5/-GTGTTGCAGATAATGAGGGC-3/ (forward), 5/- AAGGTCCTGAGGGCAAAGAG-3/ (reverse); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5/-AAGTTCAACGGCACAGTCAAGG-3/(forward); 5/-CATACTCAGCACCAGCATCACC-3/ (reverse). The levels of mRNA were normalized relative to the amount of GAPDH mRNA. The relative expression of studied genes was calculated using 2-ΔCt method [23 (link)].
10
Liver RNA Extraction Protocol
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Liver samples were homogenized in phosphate buffer (pH 7.0) at 4 °C with homogenizer (Ma et al., 2017 (link)). Total RNAs were extracted from liver tissues of all rats using the RNX-Plus (SinaClon; RN7713C) Kit. Nanodrop ND-1000 spectrophotometer (Thermo Sci., Newington, NH) method was applied to estimate the quantity and quality of extracted RNAs.
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