Total RNA was extracted from the BNYVV-infected plants using RNX-Plus kit (Sinaclon, Tehran, Iran) followed by DNase treatment. cDNA was synthesized using first strand cDNA synthesis kit (Sinaclon, Tehran, Iran). RT-PCR reaction was done by the first cycle at 93 °C for 6 min, being followed by 35 cycles at 93 °C for 30 sec, 65 °C for 45 sec, 72 °C for 1 min and a last cycle at 72 °C for 10 min. Then, the PCR products were visualized by agarose gel electrophoresis.
First strand cdna synthesis kit
Manufactured by Sinaclon
The First Strand cDNA Synthesis Kit is a laboratory tool designed for the reverse transcription of RNA into complementary DNA (cDNA). It provides the necessary reagents and enzymes required to generate first-strand cDNA from various RNA templates, which can then be used for downstream applications such as PCR, RT-PCR, and gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rnx plus kit, by Sinaclon (2 mentions)
Curcumin, by Santa Cruz Biotechnology (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Dmso dimethyl sulfoxide, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Rnx plus solution, by Sinaclon (1 mentions)
4 protocols using first strand cdna synthesis kit
1
BNYVV RNA Extraction and RT-PCR Analysis
2
Viral RNA Extraction and cDNA Synthesis
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According to the manufacturer's instructions, viral RNA was extracted from all samples using the RNX-PLUS kit (Sinaclon, Iran). In brief, 1 ml of RNX-PLUS solution and 200 μl of chloroform were added to the tube containing the sample. Then, the mixture was centrifuged at 12000g at 4°C for 15 min. Next, the aqueous phase was transferred to a new tube, and an equal volume of isopropanol was added. The supernatant was discarded after centrifugation, and 1 ml of 75% ethanol was added to the mixture. Afterward, the supernatant was removed, and the sediment was dried at room temperature. The concentration of the RNAs extracted was measured using a spectrophotometer. Following this, complementary DNA (cDNA) synthesis was performed using SinaClon first-strand cDNA synthesis kit according to manufacturer's instructions.
3
Curcumin Cytotoxicity Assay in Cells
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Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) was purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide, a yellow tetrazole], DMSO (dimethyl sulfoxide), RNase, and propidium iodide were obtained from Sigma (Sigma, St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and trypsin/EDTA were purchased from Gibco (Pittsburgh, PA, USA). RNX-Plus Solution and the First Strand cDNA Synthesis Kit were purchased from Sinaclon (Sinaclon, Tehran, Iran). Finally, 5-fluorouracil (5-FU) was provided by EBEWE PHARMA (EBEWE Pharma, Unterach, Austria).
4
Quantifying miRNA-601 and PD-L1 Levels
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RNA from the samples was extracted using a First Strand cDNA Synthesis kit (SinaClon Co.,
Iran) according to the manufacturer’s instructions. Real-time PCR was performed for
miRNA-601 and PD-L1, as the targets, and β-actin, as the
reference gene. Table 1 lists the primers used for these genes.
A total volume of the 20 µl PCR reaction, which included 4 µl distilled water, 10 µl
master mix, 4 µl cDNA, and 1 µl primers was used. The amplification program was designed
according to the appropriate annealing temperature: 1 cycle at 95°C for 60 seconds,
followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 61°C for 40
seconds, and 72°C for 45 seconds. Expression of the target miRNA-601 was
normalized by miRNA REST software. No miRNA was used as the negative control and all tests
were performed in triplicate.
Iran) according to the manufacturer’s instructions. Real-time PCR was performed for
miRNA-601 and PD-L1, as the targets, and β-actin, as the
reference gene. Table 1 lists the primers used for these genes.
A total volume of the 20 µl PCR reaction, which included 4 µl distilled water, 10 µl
master mix, 4 µl cDNA, and 1 µl primers was used. The amplification program was designed
according to the appropriate annealing temperature: 1 cycle at 95°C for 60 seconds,
followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 61°C for 40
seconds, and 72°C for 45 seconds. Expression of the target miRNA-601 was
normalized by miRNA REST software. No miRNA was used as the negative control and all tests
were performed in triplicate.
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