RNA was extracted immediately after sampling using total RNA extraction kit (Yekta Tajhiz Azma, Iran) according to the manufacturer's instruction. For each sample, the concentration of RNA was measured using spectrophotometer; overall, the absorption 260/280 ratio for all samples was >1.8. Extracted RNA was stored at −80°C. After that, 4 μg of each RNA sample was treated by 1 U of DNase I RNase-free (1 U/μl) (Sinaclon, Iran). In the next step, complementary DNA (cDNA) of each sample was synthesized using cDNA synthesis kit (BioFACT, 2X RT Pre-Mix, South Korea). For cDNA synthesis, 2 μg of DNase I treated RNA was reverse transcribed in a total volume of 20 μl containing 10 μl 2X RT Pre-Mix and 50 μM oligo dT.
Total rna extraction kit
Manufactured by Yekta Tajhiz Azma
The Total RNA Extraction Kit is designed for the rapid and efficient extraction of high-quality total RNA from a variety of biological samples. The kit utilizes a specialized lysis buffer and column-based purification method to isolate intact RNA suitable for downstream applications such as RT-qPCR, RNA sequencing, and Northern blotting.
Automatically generated - may contain errors
3 protocols using total rna extraction kit
1
RNA Extraction and cDNA Synthesis
2
Quantitative Analysis of Autophagy Genes in THP-1 Cells
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Total RNA was extracted from THP-1 cell line in each tested well using total RNA extraction kit (Yekta Tajhiz Azma, Tehran, Iran) in accordance with the manufacturer’s protocol [37 (link)]. Extracted RNA was purified by DNase (Thermo Fisher Scientific) treatment, and its concentration was calculated by NanoDrop (NanoDrop Technologies, USA). After adjustment complementary DNA (cDNA) was synthesized using cDNA synthesis kit (Yekta Tajhiz Azma, Tehran, Iran).
To analyze the expression of levels of atg5, atg7, atg12, lc3b, and β-actin (BACT) genes, amplification of corresponded genes was performed using Rotor-Gene Q (Qiagen, Germany) thermocycler. The reaction mixture of 20 µl contained 1 µL of each cDNA sample, 10 µl SYBR green qPCR master mix 2X (Ampliqon, Denmark), and 0.5 µL of each primer [37 (link)]. The final volume was adjusted by adding RNase/DNase-free water. The thermal cycling conditions consisted of an initial denaturation of 10 min at 95℃ followed by 40 cycles of 95℃ for 20 s, 59–61 °C for 30 s, 72℃ for 20 s and a final extension step at 72℃ for 20 Sect. [37 (link)]. The melt curve analysis was performed for each gene to rule out nonspecific amplifications. Relative expression level of each gene was compared to the β-actin gene, and results were analyzed using the 2− ΔΔCt method incorporated into the relative expression software (REST).
To analyze the expression of levels of atg5, atg7, atg12, lc3b, and β-actin (BACT) genes, amplification of corresponded genes was performed using Rotor-Gene Q (Qiagen, Germany) thermocycler. The reaction mixture of 20 µl contained 1 µL of each cDNA sample, 10 µl SYBR green qPCR master mix 2X (Ampliqon, Denmark), and 0.5 µL of each primer [37 (link)]. The final volume was adjusted by adding RNase/DNase-free water. The thermal cycling conditions consisted of an initial denaturation of 10 min at 95℃ followed by 40 cycles of 95℃ for 20 s, 59–61 °C for 30 s, 72℃ for 20 s and a final extension step at 72℃ for 20 Sect. [37 (link)]. The melt curve analysis was performed for each gene to rule out nonspecific amplifications. Relative expression level of each gene was compared to the β-actin gene, and results were analyzed using the 2− ΔΔCt method incorporated into the relative expression software (REST).
3
Isolation and Characterization of PBMCs
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Venous whole blood specimens (5 cc) were transferred to collection tubes with EDTA. Peripheral blood mononuclear cells (PBMCs) were isolated via lymphocyte separation media. The blood samples by Ficoll as the lymphocyte separation media, were centrifuged at 800 ×g for 20 min. Then, the PBMCs were washed twice by phosphate buffered saline (PBS), at pH 7.4. RNA was extracted from PBMCs by Total RNA Extraction Kit (Yekta tajhiz Azma, Tehran, Iran) based on the manufacturer’s instructions. The Easy™ cDNA Reverse Transcription kit (ParsTous, Iran) was used to perform reverse transcription of total RNA into cDNA. Briefly, the template RNA (total RNA or Poly (A) mRNA) and other kit components in RNase-free tube were mixed. Next, the mixture was rapidly vortexed and then, incubation was performed for 10 min at 25°C and 1 hour at 47°C, respectively. The reaction was stopped by heating at 85°C for 5 minutes. At the end, cDNA products were stored at -80°C.
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