Analysis of candidate gene expression. (a) General principles. Messenger RNAs from the different cell populations (cells A and B) are reverse transcribed. Multiplex PCR is then performed using specific primer pairs to amplify the cDNAs of interest. The resulting mixture of PCR products is radiolabeled and these complex probes are used to hybridize identical membranes spotted with the candidate gene cDNA targets. After autoradiography, the intensities of the hybridization signals are compared and quantified. Arrows indicate the positions of differentially expressed genes. The absence of hybridization (open circles) indicates that the candidate gene is not expressed. (b) Efficiency of the technique and examples of differentially expressed genes. The expression of different candidate genes was compared in either unsynchronized (UN), growth-arrested (GA), G1 phase (G1), G1/S transition (G1/S) or G2 phase (G2) cultures of Nb2 cells. The efficiency of the technique was controlled using equivalent amounts of rabbit a and ß globin cDNAs, which were included on the nylon membranes along with the candidate gene targets. The two globin cDNAs were added in different amounts (50 or 150 ng) to each cDNA population before co-amplification. For each population tested, filters were hybridized with both globin probes, but only representative hybridization signals are shown, for either a (Panel A) or ß (Panel B) globin. Numbers 1 and 3 represent the relative amount of the control rabbit globin cDNAs added, and are reflected in the differences in the intensity of the hybridization signals. Thus, a threefold difference in the quantity of a particular transcript in the initial population generates a clear difference in the intensity of the corresponding hybridization signals. Rows C, D, E and F are examples of the results obtained with ganglioside synthase GD3, EGR-1, FAK p125 and Stat3, respectively. Except for Stat3, which is not differentially expressed in probes UN, GA, G1, G1/S and G2, the three other genes showed a clear differential expression. (c) Northern blot analysis showing the constitutive expression of EGR-1 during Nb2 cell-cycle progression. Growth-arrested Nb2 cells were stimulated with ovine prolactin and collected after various periods of stimulation corresponding to different stages of the cell cycle (G1, G1/S and G2). The expression of EGR-1 was evaluated by northern blot using 10 µg of total RNA from the various times following prolactin stimulation. Ethidium bromide (EtBr) staining of the gel is shown as a control (18S and 28S rRNA).