Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

Figure 1 Schematic diagram of preparing representations for cDNA RDA based on cDNA libraries. Using BamH I alone (A) or BamH I and Xho I together (B) to digest library DNAs. Black boxes represent insert cDNAs of cDNA libraries. White boxes represent two arms of cDNA library vector (λZAP II vector). “B” epresents BamH I and “X”Xho I.

Figure 2 Agarose gel electrophoresis of digestion products of Alli823 (lanes 1, 3, 5) and BGC823 (lanes 2, 4, 6) cDNA library DNAs digested with BamH I at 37 °C for 2 (lanes 1, 2), 4 (lanes 3, 4), and 12 h (lanes 5, 6) respectively. The digestion products of Alli823 (lane 7) and BGC823 (lane 8) library DNAs digested with BamH I and Xho I together (A fragment, 850 bp in size, appeared in the digestion products). λPhage/Hind III size marker (lane M).

Figure 3 Agarose gel electrophoresis of amplicons derived from different enzyme digestions. A: The amplicons obtained by using BamH I to digest Alli823 (lane 1) and BGC823 (lane 2) cDNA library DNAs respectively; B: The amplicons obtained by using BamH I and Xho I together to digest Alli823 (lane 1) and BGC823 (lane 2) library DNAs respectively. 1 kb size marker (lane M).

Figure 4 A: Agarose gel electrophoresis of reamplified difference products SH1-4 (lanes 1-4 respectively). PCR product of GAPDH (400 bp in size) used as quantity control (lane G); B: Slot blots analysis showing differentially expressed cDNAs. Reverse transcription products (first stranded cDNA) of total RNA of BGC823 and Alli823 cells used as probe respectively; C: The degree to which each cDNA was up-regulated.

Figure 5 Sequences of two novel ESTs (SH1 and SH4) isolated by cDNA RDA based on cDNA libraries.

Figure 6 Northern blotting result showing the expression level of calcyclin gene in human gastric cancer cell lines BGC823, MGC803, PAMC82, SGC7901 and MKN45 (lanes 1-5) respectively. The arrow showing the novel transcript derived from the recombinant gene merged from calcyclin gene and HNRPA0 gene.