Mrna extraction kit

BEAS-2B cells were seeded in a 6-well plate at a density of 3 × 10⁵ cells per well overnight and then pre-treated with 0.1 and 20 μM of SAHA for 24 h, followed by 1 μg/mL of LPS treatment for 24 h. mRNA was extracted using the mRNA extraction kit from Thermo Fisher Scientific. Then, 0.5 μg of mRNA sample was used to synthesize cDNA by adding SuperScript III First-Strand cDNA Synthesis System purchased from Invitrogen, NY, USA. The cDNA was used as the template for real-time PCR using the SYBR Green PCR master mix (Applied Biosystem, CA, USA) to measure the RNA expression of various genes. The mRNA expression was calculated as the fold change with normalization to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the 2−ΔΔCT method, while GAPDH was used as an internal loading control. Primers were designed using Primer-Blast software and purchased from Integrated DNA Technologies (IDT, Coralville, Iowa, USA). Primer sequences are listed in Supplementary Table S1. Three independent experiments confirmed the level of mRNA expression.

HCT116 cells were plated in 6-well plate at a density of 3 x 10⁵ cells/well overnight and then treated with either vehicle control, 1 mM, 5 mM and 10mM NaB for 24h. mRNA was extracted using mRNA extraction kit from Thermo Fisher Scientific (Cat No. K0732). The first-strand cDNA was synthesized from 1 μg extracted RNA using SuperScript III First-Strand cDNA Synthesis System (Invitrogen, Grand Island, NY, USA). To determine the RNA expression of specific genes, the cDNA was used as the template for real time PCR using Power SYBR Green PCR Master Mix (Applied Biosystem, Carlsbads, CA). The mRNA expression was calculated as the fold change with normalization to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the 2^−ΔΔCT method while GAPDH was used as an internal loading control. The primers were designed and ordered from Integrated DNA Technologies (IDT, Coralville, Iowa, USA) (Supplemental Table 1).