Both neutral and acidic SMase mRNAs are expressed by articular cartilage chondrocytes. Following 24 hours of culture, cells were placed into TRIZOL® (1 × 106 cells/ml) and total RNA extracted, in accordance with the manufacturer's instructions. cDNA was generated (n = 4) and PCR performed using primers specific to GAPDH, or acidic or neutral SMase (Table 1). Amplified products were separated alongside a 100 bp DNA ladder (L) on 1–2% agarose gels, containing ethidium bromide (10 μg/ml). Product sizes (bp) are indicated. bp, base pairs; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; SMase, sphingomyelinase.
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