Semiquantitive PCR is a method to evaluate expression of target genes by analyzing RT-PCR products in comparison to standards.Two measures are involved;one is that primers for target gene and internal standard are put...Semiquantitive PCR is a method to evaluate expression of target genes by analyzing RT-PCR products in comparison to standards.Two measures are involved;one is that primers for target gene and internal standard are put into the same tube to run PCR,and the other one is that primers for target gene and internal standard run PCR in separate tubes.In research of environmental endocrine disruption semiquantitive PCR is a usual means to measure estrogen-like effects which result from endocrine disruptors.In order to discuss advantages and disadvantages of these two methods for further research on endocrine disruption in Carassius auratus,RT-PCR was used to evaluate of expression of 776 bp of estrogen receptor gene(ER gene) in liver after taking the injection of E2 in C.auratus by following these two methods respectively,with 18S as internal standard.The results showed that ER gene and 18S gene were amplified successfully following both methods.The first method-putting primers for different gene in the same tube to run PCR might contain problems such as competition and different plat phase between target gene and internal standard,and it is needed to grope appropriate ratio of primers and numbers of cycles,yet it is more accurate.And another method putting primers for different genes in separate tubes to run PCR is easier because of the absence of no problemsexisted in the first method,whereas it is less accurate than the first one.In addition,based on the results,18S is an ideal internal standard for both methods in the research of estrogen-like effects in C.auratus.展开更多
The field-amplified sample injection behavior of cationic tripropylamine(TPA) and anionic proline(Pro) at a positive voltage in capillary electrophoresis with tris(2,2′-bipyridyl)ruthenium(Ⅱ) electrochemiluminescenc...The field-amplified sample injection behavior of cationic tripropylamine(TPA) and anionic proline(Pro) at a positive voltage in capillary electrophoresis with tris(2,2′-bipyridyl)ruthenium(Ⅱ) electrochemiluminescence(ECL) detection system was studied. In the case of TPA, where the sample solution was prepared in pure water, ECL sensitivity can be improved by 100 times compared to conventional electroinjection method when a positive voltage was applied. Under the same positive voltage condition, anionic Pro prepared in electrolyte solution can also be injected and concentrated in the column when a water plug was injected before sample introduction. The sensitivity and efficiency were enhanced by 10 and 46 times, respectively. The behavior of cationic TPA can be explained by conventional field amplified sample injection(FASI)theory. When the ratio of resistivities of sample matrix to that of separation buffer is less than 1(γ1), the conventional FASI theory can also be used to explain the improved sensitivity and theoretical plates of Pro. The sensitivity, plate, velocity(v ep), amplified factor(v ep/v 0 ep) and peak variance(σ 2) of Pro reach maximum at optimized water plug length and buffer concentration of the sample matrix.展开更多
文摘Semiquantitive PCR is a method to evaluate expression of target genes by analyzing RT-PCR products in comparison to standards.Two measures are involved;one is that primers for target gene and internal standard are put into the same tube to run PCR,and the other one is that primers for target gene and internal standard run PCR in separate tubes.In research of environmental endocrine disruption semiquantitive PCR is a usual means to measure estrogen-like effects which result from endocrine disruptors.In order to discuss advantages and disadvantages of these two methods for further research on endocrine disruption in Carassius auratus,RT-PCR was used to evaluate of expression of 776 bp of estrogen receptor gene(ER gene) in liver after taking the injection of E2 in C.auratus by following these two methods respectively,with 18S as internal standard.The results showed that ER gene and 18S gene were amplified successfully following both methods.The first method-putting primers for different gene in the same tube to run PCR might contain problems such as competition and different plat phase between target gene and internal standard,and it is needed to grope appropriate ratio of primers and numbers of cycles,yet it is more accurate.And another method putting primers for different genes in separate tubes to run PCR is easier because of the absence of no problemsexisted in the first method,whereas it is less accurate than the first one.In addition,based on the results,18S is an ideal internal standard for both methods in the research of estrogen-like effects in C.auratus.
文摘The field-amplified sample injection behavior of cationic tripropylamine(TPA) and anionic proline(Pro) at a positive voltage in capillary electrophoresis with tris(2,2′-bipyridyl)ruthenium(Ⅱ) electrochemiluminescence(ECL) detection system was studied. In the case of TPA, where the sample solution was prepared in pure water, ECL sensitivity can be improved by 100 times compared to conventional electroinjection method when a positive voltage was applied. Under the same positive voltage condition, anionic Pro prepared in electrolyte solution can also be injected and concentrated in the column when a water plug was injected before sample introduction. The sensitivity and efficiency were enhanced by 10 and 46 times, respectively. The behavior of cationic TPA can be explained by conventional field amplified sample injection(FASI)theory. When the ratio of resistivities of sample matrix to that of separation buffer is less than 1(γ1), the conventional FASI theory can also be used to explain the improved sensitivity and theoretical plates of Pro. The sensitivity, plate, velocity(v ep), amplified factor(v ep/v 0 ep) and peak variance(σ 2) of Pro reach maximum at optimized water plug length and buffer concentration of the sample matrix.
