Rapid method for relative gene expression determination in human tissues using automated capillary gel electrophoresis and multicolor detection.
The aim of this study was to evaluate a direct and automated post-polymerase chain reaction (PCR) detection system to simultaneously determine the relative gene expression levels of nine cancer-related human genes. Total RNA was prepared from flash-frozen biopsies derived from human colorectal tumors or normal mucosa and reverse-transcribed to cDNA which was PCR-amplified using primer pairs corresponding to the studied genes. In each reaction, the forward primer was labeled with a fluorescent dye. The PCR products were pooled and an internal size standard with a uniquely colored fluorescent dye was added. The samples were then subjected to automated capillary gel electrophoresis. Fragment analysis software was used to calculate the relative gene expression using beta-actin as the reference gene. We found that automated capillary gel electrophoresis with multicolor detection is a rapid, accurate and highly reproducible method for separation and quantification of PCR-amplified cDNA.
Odin
E
E
Department of Surgery, Sahlgrenska University Hospital/Ostra, Gothenburg, Sweden.
Wettergren
Y
Y
Larsson
L
L
Larsson
P A
PA
Gustavsson
B
B
D001322:Autoanalysis; D003106:Colon / Q000737:chemistry; D015179:Colorectal Neoplasms / Q000235:genetics*; D018076:DNA, Complementary / Q000032:analysis; D004273:DNA, Neoplasm / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D015870:Gene Expression; D006801:Humans; D007413:Intestinal Mucosa / Q000737:chemistry; D012007:Rectum / Q000737:chemistry; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013940:Thymidylate Synthase / Q000235:genetics
D018076:DNA, Complementary; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D013940:Thymidylate Synthase