Rneasy mrna extraction kit

Passage 1 hAF cells were seeded at a seeding density of 30 000 cells/well and cultured to 70% to 80% confluency in six‐well cell culture plates in Ham's F‐12 medium supplemented with 10% FBS and 1% P/S (2 mL media/well). A low cell density was used for seeding and the cells allowed to expand before experimentation as the number of cells obtained from the human tissue was low due to the tissue amount procured. Cells were treated with fresh media containing 0, 0.5, and 1.0 mM polyP‐22. After 48 hour, hAF cells were washed with phosphate‐buffered saline (PBS−/−, calcium and magnesium free) and then lysed with RLT lysis buffer (Qiagen, Valencia, California) and RNA was isolated using RNeasy mRNA extraction kit (Qiagen Germantown, Maryland). Quantitative RT‐PCR was performed to measure relative gene expression of anabolic and catabolic genes of interest as previously described.³² Sequence specific primers were used for aggrecan (ACAN), collagen type I (Col1A1), and matrix metalloproteinases 1 and 3 (MMP‐1 and ‐3) (Table 2). After normalization with GAPDH expression levels, the relative gene expression was reported as relative fold expression compared to control, using the ΔΔCt method.³⁸