Total RNA was extracted from the serum of patients by RiboEX LS (GeneAll, Seoul). 600 μL RiboEX LS Reagent was added to 200 μL serum, and was incubated for 5 minutes at room temperature (25°C), then, 160 μL of chloroform was added and vigorously shaken, and the samples were centrifuged at 12000 g for 15 minutes at 4°C. Furthermore, supernatants were mixed with 400 μL of isopropanol and incubated overnight at −20°C. After centrifuging at 12000 g for 1 hour at 4°C, the resulting pellets were washed with 75% ethanol and centrifuged at 7500g for 5 minutes at 4°C. After allowing the ethanol to completely remove, the pellets were dissolved in 25 μL of diethylenepyrocarbonate-treated water (DEPC-treated water) (SinaClon. Iran). The quality/quantity and purity of RNA was analyzed by optical density measurement with NanoDrop.
Depc treated water
Manufactured by Sinaclon
DEPC-treated water is a laboratory-grade water that has been treated with diethylpyrocarbonate (DEPC) to inactivate RNase enzymes. It is commonly used in molecular biology applications to ensure the integrity of RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (3 mentions)
Random hexamer primer, by Sinaclon (3 mentions)
Dntp mix, by Sinaclon (3 mentions)
Riboex ls, by GeneAll (1 mentions)
Cinnapure rna extraction kit, by Sinaclon (1 mentions)
Peqgold rna trifast, by Avantor (1 mentions)
5 protocols using depc treated water
1
Serum RNA Extraction Protocol
2
RNA Extraction and cDNA Synthesis Protocol
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RNA was extracted from tissue samples using the CinnaPure RNA Extraction Kit (SinaClon, Iran). For cDNA synthesis, 1 μl (0.2 μg) of random hexamer primer (SinaClon, Iran) was added to 5 μl of extracted RNA, and the mixture was heated at 65°C for 5 min. 14 μl of cDNA master mix containing 7.25 μl of DEPC‐treated water (SinaClon, Iran), 2 μl of dNTP mix (SinaClon, Iran), 0.25 μl of RiboLock RNase inhibitor (Thermo Fisher Scientific, USA), 0.5 μl of Revert Aid Reverse Transcriptase (Thermo Fisher Scientific, USA) and 4 μl of 5X RT reaction buffer were added to each tube, resulting in a final volume of 20 μl. The mixture was then incubated at 25°C for 5 min, 42°C for 60 min, 95°C for 5 min and 4°C for 1 min. The cDNA was stored at −20°C until use.
3
RNA Extraction and cDNA Synthesis Protocol
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RNA was extracted from tissue samples using CinnaPure RNA Extraction Kit (SinaClone, Iran). For cDNA synthesis, 1 µL (0.2 µg) of random hexamer primer (SinaClon, Iran) was added to 5 µL of extracted RNA, and the mixture was heated at 65 °C for 5 minutes. Fourteen µL of cDNA master mix containing 7.25 µL of DEPC-treated water (SinaClon, Iran), 2 µL of dNTP mix (SinaClon, Iran), 0.25 µL of RiboLock RNase Inhibitor (Thermo Fisher Scientific, USA), 0.5 µL of Revert Aid Reverse Transcriptase (Thermo Fisher Scientific, USA), and 4 µL of 5X RT reaction buffer was added to each tube, resulting in a final volume of 20 µL. Then, the mixture was incubated at 25 °C for 5 min, 42 °C for 60 min, 95 °C for 5 min, and 4 °C for 1 min. The cDNA was stored at −20 °C until use. Sampling, RNA extraction, and cDNA synthesis were conducted in Iraq, and the remainder of the work was carried out in Iran.
4
Random Hexamer-Based cDNA Synthesis Protocol
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For cDNA synthesis, 1 μL (0.2 ug) of random hexamer primer (SinaClon, Iran) was added to 5 μL of extracted RNA and the mixture was heated at 65°C for 5 minutes. Fourteen μL of cDNA master mix containing 7.25 μL DEPC-treated water (SinaClon, Iran), 2 μL dNTP mix (SinaClon, Iran), 0.25 μL RiboLock RNase Inhibitor (Thermo Fisher Scientific, USA), 0.5 μL Revert Aid Reverse Transcriptase (Thermo Fisher Scientific, USA), and 4 μL 5X RT Reaction Buffer was added to each tube, resulting in a final volume of 20 μL. Then, the mixture was incubated at 25°C for 5 min, 42°C for 60 min, 95°C for 5 min, and 4°C for 1 min, respectively. The cDNA was stored at −20°C until use (10 (link)).
5
Gene Expression Analysis of NLRP3 Inflammasome
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After sampling, the expression of NLRP3, ASC, caspase-1, Bax and Bcl2 genes in all groups was studied by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Total RNA was extracted using peqGold RNA TriFast (PeqLab, Germany) according to the manufacturer’s instructions. The RNA pellet was dissolved in diethylpyrocarbonate-treated water (DEPC treated water; SinaClon, Iran) and quantified spectrophotometrically at 260 nmwavelength. The integrity of the extracted total RNA was assessed by agarose gel electrophoresis and verified by the presence of the 28S and 18S rRNA bands. Immediately after RNA preparation, 2 μg of total RNA was used for cDNA synthesis in a total volume of 20 μl by using RevertAid ™ First Strand cDNA Synthesis Kit (Aryatous, Iran). The cDNA was stored at -80°C until use.
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