QUANTITATIVE METHOD FOR EVALUATING GENE EXPRESSION USING MULTIPLEX CONCURRENT REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION AND DEVICE FOR QUANTITATIVELY DETERMINING EXPRESSION LEVEL OF TARGET GENES

摘要

FIELD: biological engineering. SUBSTANCE: method involves reversibly transcribing and specifically amplifying genes in presence of competitive matrices. Relation of each target gene to each housekeeping gene is obtained. The relation is used for quantitatively evaluating expression of each gene. The same evaluation method is applicable in tissues. The device has flasks containing A, B and C solutions. A-solution in A flask is stock solution of reaction mixture usable for polymerase chain reaction. It has dNTPs, buffer substance, electrophoretic dye, and primers for b-actin or GAPDH (primers are optional). Capillary tube passes through sealing ring in A flask cover and then through on/off valve and enters Y-valve 1. The other capillary tube entering Y- valve 1 comes from multi- channel connector unit 1. A lot of capillary tubes entering multi-channel connector unit 1 come through on/off valve, capillary tubes entering on/off valves exit through sealing rings in covers of flasks containing separated mixtures of competitive matrices (B solutions). Four groups of competitive matrix mixtures are available (each group has 100 different competitive matrices in five different molarities of (10E^-12-10M^-16) to enable one to change groups of competitive matrix mixtures depending on necessity. Capillary coming from Y-valve 1 enters Y-valve 2, the other capillary entering Y-valve 2 comes from multi-channel connector unit 2, capillary coming from Y- valve 2 enters Y-valve 4, the other capillary entering Y-valve 4 comes from multi- channel connector unit 3, capillary coming from Y-valve 4 enters Y-valve 5. A lot of capillary tubes entering multi-channel connector unit 3 come from glass flasks each containing primer solutions (C solutions) for particular gene. On/off valves are available between Y-valve 3 and the multi-channel connector unit. The other capillaries coming from Y-valves 3 enter Y-valves 7. Capillary tube exiting from each Y-valve 3 passes through on/off valve and joins Y-valve 7. Other tubes come to Y-valve 7 from multi-channel connector unit 4 through on/off valves. Capillary entering multi-channel connector unit 4 branches from Y-valve 5. One fourth part of tubes branching from Y-valves 7 enter Y-valves 8. Other tubes entering Y-valves 8 come from Y-valves 7 through on/off valves. Tubes belonging to the rest three quarters of Y-valves 7 and tubes from Y-valves 8 join separate glass capillaries in air thermocycler unit. EFFECT: high accuracy in analyzing small samples of tissues and cells. 28 cl, 7 dwg, 1 tbl