title: Semi-quantitative detection of gene expression using  bisbenzimide dye.
abstract: An  electrochemical biosensor, using a disposable electrochemical printed chip  aggregation by the bisbenzimide dye (Hoechst 33258), was used for detecting the  expression of β-actin and RAGE genes. Using linear sweep voltammetry, the  expression of these two genes in HeLa and HepG2 cell lines was determined based  on anodic peak current, and the results were compared with conventional agarose  gel electrophoresis. Total cellular RNA was reverse transcribed to  complementary DNA, and amplification by PCR was carried out. Subsequently, the  PCR products were subjected to detection by either electrophoresis or  electrochemical biosensor. Precision of the electrochemical biosensor technique  was acceptable (β-actin: CV = 1.875% for 10(4) copies  and 4.684% for 10(9) copies; RAGE: CV = 2.253% for 10(9) copies,  and 3.743% for 10 copies). In the electrochemical biosensor technique, the PCR  products were measured in the same run with various concentrations of  standards, and copy numbers of β-actin gene were interpolated from a standard  curve. Copy numbers of the β-actin gene were then compared between the two  techniques. At the 95% confidence limit, the two methods had no significant  differences and were significantly correlated (y = -40383.0623 + 1.0233x; P  > 0.10). The electrochemical biosensor method was more sensitive than the  conventional electrophoresis method because it could detect as low as 10 copies  of the RAGE gene. The conventional electrophoresis method detected the RAGE  gene at concentrations of at least 10(4) copies,  and the linearity for semi-quantitative measurement was in the range of 10(6)-10(9) copies.  When the electrochemical biosensor was applied to detect the RAGE gene  expression in both cell types, we found that HeLa cells expressed the RAGE gene  about 2-fold higher than in HepG2 cells (relative value of 0.000905 vs 0.0004670).  Therefore, this study suggests the potential modification of the  electrochemical biosensor with the use of bisbenzimide dye (Hoechst 33258) for  detecting gene expression.
authors.affiliation: Graduate Program in Clinical Biochemistry and Molecular Medicine, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.
authors.forename: P
authors.initials: P
authors.lastname: Kittimongkolsuk
authors.forename: T
authors.initials: T
authors.lastname: Tencomnao
authors.forename: R
authors.initials: R
authors.lastname: Santiyanont
mesh_terms: D000199:Actins / Q000235:genetics; D015374:Biosensing Techniques; D006690:Bisbenzimidazole / Q000378:metabolism*; D004396:Coloring Agents / Q000378:metabolism*; D055664:Electrochemical Techniques / Q000379:methods*; D004566:Electrodes; D004587:Electrophoresis, Agar Gel; D018628:Gene Dosage / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D016133:Polymerase Chain Reaction; D000067759:Receptor for Advanced Glycation End Products / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards
chemical_list: D000199:Actins; D004396:Coloring Agents; D000067759:Receptor for Advanced Glycation End Products; D006690:Bisbenzimidazole