Leaf tissue was placed in a mortar containing liquid nitrogen and homogenized with a pestle. Approximately 100 mg of the powder was transferred to a 1.5-µL microtube. Total RNA was isolated using a Ribospin Plant RNA isolation kit (Geneall Biotechnology Co. Ltd., Seoul, Korea) as per the protocol provided by the manufacturer. RNA integrity was determined by the RNA Integrity Number, which was calculated using an Agilent 2100 Bioanalyzer (Agilent Technologies Korea Ltd., Seoul, Korea). The samples with RNA Integrity Number values greater than eight were selected for further analysis. Using total RNA as the template, cDNAs were synthesized using Geneall 2X HyperScript reverse transcription polymerase chain reaction (RT-PCR) master mix with oligo dT (Geneall Biotechnology Co. Ltd.) as per the protocol provided by the manufacturer. Reaction details for the RT-PCR are as follows: Preheating of total RNA at 65°C for 5 min followed by heating the reaction mixture to 55°C for 60 min and 95°C for 5 min.
Oligo dt
Manufactured by GeneAll
Oligo dT is a laboratory equipment used for the purification and isolation of messenger RNA (mRNA) from total RNA samples. It is a short synthetic DNA oligonucleotide with a sequence of thymine (T) nucleotides that binds to the poly(A) tail of mRNA molecules, allowing for their selective capture and separation from other RNA species.
Automatically generated - may contain errors
2 protocols using oligo dt
1
RNA Extraction and cDNA Synthesis from Leaf Tissue
2
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated with SV Total RNA Isolation System (Promega). RNA concentration was evaluated by spectrophotometric reading at 280 and 260 nm. Total RNA was used for first strand cDNA synthesis with HyperScript, First strand Synthesis Kit and Oligo-dT, as random primer (GeneAll). The PCR was performed with about 150 ng of cDNA using DreamTaq. The following primer sequences were used for amplification: Actin forward 5'-CCTTCCTGGGCATGGAGTCCTG-3', Actin reverse 5'-GGAGCAATGATCTTGATCTTC-3' (208 bp); Bax forward 5'-GCAGGGAGGATGGCTGGGGAG-3', Bax reverse 5'-TCCAGACAAGCAGCCGCTCACG-3' (352 bp).
The experimental protocols for PCR reactions were: initial denaturation for 5 min at 95°C; amplification for 40 cycles of denaturation, 30 sec, 95°C, annealing: 30 s, at 55°C (Actin), 60°C (Bax) and elongation at 72°C for 1 min; final elongation for 10 min at 72°C. PCR products were then analysed by 1.5 % agarose gels electrophoresis in TBE1X Buffer. Image acquisition and product analysis was made by Bio-Rad imaging systems with Quantity One1-D analysis software. The density of the PCR bands were divided by that of the housekeeping gene (Actin) and expressed as percent of the control band density.
The experimental protocols for PCR reactions were: initial denaturation for 5 min at 95°C; amplification for 40 cycles of denaturation, 30 sec, 95°C, annealing: 30 s, at 55°C (Actin), 60°C (Bax) and elongation at 72°C for 1 min; final elongation for 10 min at 72°C. PCR products were then analysed by 1.5 % agarose gels electrophoresis in TBE1X Buffer. Image acquisition and product analysis was made by Bio-Rad imaging systems with Quantity One1-D analysis software. The density of the PCR bands were divided by that of the housekeeping gene (Actin) and expressed as percent of the control band density.
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