RT-PCR amplicons from c-fos and fosB transcripts. A) Schematic drawing of c-fos and fosB gene structures. Exons (E) are boxed and shaded in grey. Introns (I) are indicated by lines between exons, except for the alternatively spliced sequences that are indicated by brick filled in boxes. The beginning and end of exons are indicated by the corresponding nucleotide positions (NCBI/GeneBank accession numbers: V00727 and AF093624 for c-fos and fosB, respectively). B) Names, sequences and 5'-position of upper (U) and lower (L) primers. C) Agarose (1.5%) gel electrophoresis analysis of RT-PCR products generated by different primer pairs (identified at the top of each line). Total RNA from NIH 3T3 cells was used as template. Forty cycles of PCR were performed as detailed . Genomic DNA was further amplified by the E1U-E2L primer pair to exclude the possibility that our PCR conditions were not optimal for the amplification of the longest theoretical fragment, i.e. the 933 nt amplicon that should be observed if intron 1 remained unspliced in the c-fos transcript population [see Additional file 7]. The molecular weight marker was a 100-nt ladder from Roche.