Application of fluorescently labeled poly(dU) for gene expression profiling on cDNA microarrays.

The membrane filter hybridization technique has been widely used for gene expression profiling. The preparation of sensitive and reliable probes is critical for quantitative analysis in this technique. We report a method in which fluorescently labeled poly(dU) is used to detect poly(A)-containing mRNA that hybridizes to specific gene targets. The probe can be used commonly for every sample, alleviating problems encountered in preparing cDNA probes by reverse transcription, particularly when many samples are to be analyzed. Moreover, the sensitivity is at least comparable to cDNA probes prepared by conventional protocols, and intensities of signals after hybridization are independent of mRNA sizes and solely dependent on copy numbers. This method was also shown to be applicable to DNA chip technology.

Osteoclast differentiation factor modulates cell cycle machinery and causes a delay in s phase progression in RAW264 cells.

Osteoclast differentiation factor (ODF) induces differentiation of mouse RAW264 cells to mature osteoclasts. To understand the mechanism controlling a coupling between withdrawal from the cell cycle and differentiation, we examined cell cycle progression and expression profiles of cell cycle regulatory genes at the initial phase in committed cells. ODF rapidly converted the hyperphosphorylated form of the retinoblastoma protein (pRb) into the hypophosphorylated form. The p21 protein was induced by ODF treatment in the same time course with that of dephosphorylation of pRb, followed by a sharp decline. After this period, a delayed entry of the S phase started accompanying the induction of CycD3 and cdk6 in differentiating cells. Hydroxyurea treatment indicated that the S phase entry was a prerequisite for osteoclast formation. Thus, ODF induces pleiotropic effects on cell cycle regulatory genes in RAW264 cells during the initial phase of the differentiation process to osteoclasts.