Figure 4

RT-PCR of 20 gene candidates. Lane 1: low molecular weight DNA ladder from New England Biolabs. Size range: 25 bp to 766 bp. Lane 2-21: 20 gene candidates, VBISMa0080, VBISMa0492, VBISMa1337, VBISMb0078, VBISMb0839, VBISMc0095, VBISMc0802, VBISMc1000, VBISMc1221, VBISMc1492, VBISMc1793, VBISMc2171, VBISMc2174, VBISMc2596, VBISMc2940, VBISMc2955, VBISMc3188, VBISMc3282, VBISMc4046 and VBISMc4289, respectively. For majority RT-PCR reactions, each produced one corresponding PCR product (lane 3-5, 7, 9-12, 14-19 and 21). These PCR products were directly sequenced and their sequences matched to the corresponding gene candidates. Multiple PCR products were found in lane 2, 6, 8, 13 and 20. The bands with the correct PCR product sizes are labeled with *. These PCR products were used to do a second round of PCR to produce enough DNA for sequencing. The sequencing results confirmed that they matched to the corresponding gene candidates. The most abundant PCR product in lane 2 was sequenced and determined to be a part of 23s rRNA sequence. Lane 22-29: negative controls using the RNA sample that was not reverse transcribed and primer pairs of new genes to show no genomic DNA contamination. In each lane of 22 to 29, combined primer pairs of two or three genes were used. Lane 30: no template control. Primer pairs of cm0012a, cm012b and cm016a, cm016b were used.