Construction and selection of subtracted cDNA library of mouse hepatocarcinoma cell lines with different lymphatic metastasis potential

Figure 1 Total RNA from Hca-F and Hca-P cells. Lane 1: RNA from Hca-F cells, Lane 2: RNA from Hca-P cells.

Figure 2 Results of secondary PCR amplification. Lanes 1-3: Product of primary PCR amplification, Lane 4: Secondary PCR amplification product of PCR control cDNA, Lane 5: Second-ary PCR amplification product of unsubtracted cDNA ,Lane 6: Secondary PCR amplification product of subtracted cDNA.

Figure 3 Analysis of subtraction effect. PCR was performed on subtracted (Lanes 1-4) or unsubtracted (Lanes 5-8) second-ary PCR product with G3PDH 5’¯Primer and 3’¯primer. Lanes 1, 5: 20 cycles, Lanes 2, 6: 25 cycles, Lanes 3, 7: 30 cycles, Lanes 4, 8: 35 cycles.

Figure 4 Results of clone PCR amplication. There was a aver-age insert size of 0.1-0.7 kb.

Figure 5 Sequence of clone 10-3

Figure 6 Gene structure of clone 10-3. Both nested primer 1 and nested primer 2R in the cDNA sequence were contained in subtracted library, indicating that only cDNA with both 2 adap-tors could be amplified by PCR.

Figure 7 Result of clone 10-3 sequencing.