Total RNA was isolated from cells using the Paris Total RNA Isolation Kit (Ambion, Carlsbad, USA) in accordance with the instructions of the manufacturer. The RNAs isolated from the hormone treated cells were converted to cDNA through a reverse transcription (Bioneer, Alameda, USA). Primer sets for the amplification of piR–651, piR–823 and Glyseraldehide-3-phosphate dehydrogenase (GAPDH) were designed and supplied by Alpha DNA, Montreal, Quebec. RT-PCR was carried out inside a Strategene MxPro3000 (Strategene, UK). GAPDH (Alpha DNA, Montreal, Quebec) was used as an internal control, and the expression of piR–651 and piR–823 was normalized in line with the expression of GAPDH. Gene expression changes were quantified using the delta-delta CT method. The forward and reverse primer sequences used in RT-PCR are shown in Table 1 [10 ,25 (link),27 (link)].
Reverse transcription
Manufactured by Bioneer
Sourced in United States
Reverse transcription is a laboratory technique that uses an enzyme called reverse transcriptase to convert single-stranded RNA into complementary DNA (cDNA). This process is a crucial step in many molecular biology applications, such as gene expression analysis, virus detection, and vaccine development.
Automatically generated - may contain errors
2 protocols using reverse transcription
1
Quantification of piRNA Expression
2
Evaluating Bcl-2 Expression in Cell Cultures
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The cells were diluted to 1 × 105 cell/mL and were incubated for 24 hours in six-well plates (Falcon, BD Biosciences). After washing the culture media twice with PBS, serum-free media was applied. H2O2 and bevacizumab were challenged at different concentrations (0, 100, 200, 300, 400 µM and 0.33, 0.67, 1.33, 2.67 mg/mL) and incubated for 16 hours. Total RNA was isolated using the total RNA purification kit (Invitrogen). Isolated RNA was quantified, and 1 µg of total RNA and 100 pmol of oligo dT were added to the reverse transcription (RT) premix (Bioneer, Seoul, Korea) to prepare 20 µL of cDNA. Preformed cDNA, Bcl-2 primer, and glycerol-3-phosphate dehydrogenase (GAPDH) primer were mixed with polymerase chain reaction (PCR) premix (Bioneer), and PCR was performed. The sequences of the primers are described in Table 1 . Next, 10 µL of each amplificate were assessed using 1.5% agarose gel electrophoresis. Quantification of Bcl-2 mRNA content was performed using computer-assisted video densitometry (Eagle Eye II-system; Stratagene, La Jolla, CA, USA).
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