Total cellular RNA was extracted using the RiboEx reagent (GeneAll) according to the manufacturer’s instructions. 1 μg of total RNA was reverse transcribed using M-MLV reverse transcriptase (ELPiS) and amplified by PCR using G-Taq polymerase (Cosmo Genetech). The following primers were used for splicing studies: Exon6.F (5′-ATAATTCCCCCACC ACCTCC-3′), Exon8.R (5′-ACTACAACACCCTTCTCACAG-3′), pcDNA.F (5′-CACTGCTTACTGGCTTATCGAA-3′), pcDNA.R (5′-CTAGAAGGCACAGTCGAGGCT-3′), AdML.F (5′-ACTCTT GGATCGGAAACCCGT-3′).
M mlv reverse transcriptase
Manufactured by Cosmogenetech
M-MLV reverse transcriptase is an enzyme used in molecular biology to synthesize complementary DNA (cDNA) from a single-stranded RNA template. It catalyzes the process of reverse transcription, converting RNA into DNA.
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2 protocols using m mlv reverse transcriptase
1
RNA Extraction and Splicing Analysis
2
Real-Time PCR Gene Expression Analysis
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Three independent replicates were conducted with two different batches of cells (A and B) at passage 6 and 7 (A passage 6, A passage 7, and B passage 6). Total RNA was extracted from pellets using the PureLinkTM RNA Mini kit (Life Technologies, Camarillo, CA, USA). The synthesis of cDNA was performed using M-MLV reverse transcriptase (Cosmogenetech, Seoul, Korea) according to the manufacturer’s instructions. Real-time PCR was performed using SYBR Pre-mix Ex TaqTM ІІ (Takara, Tokyo, Japan) and the 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). The primers used are listed in Supplementary Data (Table S3) . The PCR reaction was performed for 30 s at 95 °C, followed by 40 amplification cycles of 5 s at 95 °C and 34 s at 60 °C. The comparative CT method was used to measure the level of expression. Glyceraldehyde 3-phophate dehydrogenase (GAPDH) was used as a housekeeping gene for normalization.
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