Objective To compare the quality and quantity of total RNA from different source-original neurons applied in LMPC technique. Methods （ 1 ） Aglient 2100 bioanalyzer and RT-PCR were used to check the concentration and...Objective To compare the quality and quantity of total RNA from different source-original neurons applied in LMPC technique. Methods （ 1 ） Aglient 2100 bioanalyzer and RT-PCR were used to check the concentration and fragmentation of total RNA from unfixed, temporal fixed and fixed 12 h hypothalamus sections; （2）Different neurons of PVN and SON were collected by LMPC, CRH, TRH, AVP, OT mRNA level were measured by RT-PCR; （3）Labeled neurons by injecting CTB into stomach and non-labeled neurons in DMV collected by LMPC were checked for house keeping genes by RT-PCR. Results （ 1 ） Unfixed section had higher concentration and better quality of total RNA compared with fixed sections applied in LMPC ; relative short amplicons such as GAPDH, NSE, MCH and MCAR were successfully obtained from fixed and unfixed and long amplicon of GR can only be obtained from unfixed material; （2） In magnocellular PVN and SON the expressions of AVP and OT were more special than those in the parvocellular PVN. Oppositely, the expressions of CRH, TRH in the parvocellular were more special than the other two ; （3） The expressions of house keeping genes had no significant difference between labeled and non-labeled DMV neurons. Conclusion The quality and quantity of total RNA from unfixed brain tissues were better than fixed tissues applied in LMPC and the CTB tracer which may differentiate neurons had no significant effect on physiology of the neurons applied in LMPC. The results showed that the LMPC technique is suitable for the qualitative and quantitative study on individual neurons at mRNA level.展开更多
文摘S.tenebrarius H6主要产生安普霉素、氨甲酰妥布霉素和氨甲酰卡那霉素B,其中后两者仅在3′位有差异。为克隆相关基因,根据已报道的aprD3、livY、gentY基因设计兼并性PCR引物,并采用SON-PCR(singleoligonucleotide nested PCR)方法和LASP-PCR(linear amplification single primer PCR)方法,扩增获得部分SCD1基因,Blast分析为短链脱氢酶基因。采用基因阻断技术,使S.tenebrariusH6中SCD1基因失活。与野生菌株相比,变株的抗生素组分和含量几乎没有变化,但变株产孢子能力下降,且产孢子时间推迟,推测该基因与孢子的生成调节相关。
文摘Objective To compare the quality and quantity of total RNA from different source-original neurons applied in LMPC technique. Methods （ 1 ） Aglient 2100 bioanalyzer and RT-PCR were used to check the concentration and fragmentation of total RNA from unfixed, temporal fixed and fixed 12 h hypothalamus sections; （2）Different neurons of PVN and SON were collected by LMPC, CRH, TRH, AVP, OT mRNA level were measured by RT-PCR; （3）Labeled neurons by injecting CTB into stomach and non-labeled neurons in DMV collected by LMPC were checked for house keeping genes by RT-PCR. Results （ 1 ） Unfixed section had higher concentration and better quality of total RNA compared with fixed sections applied in LMPC ; relative short amplicons such as GAPDH, NSE, MCH and MCAR were successfully obtained from fixed and unfixed and long amplicon of GR can only be obtained from unfixed material; （2） In magnocellular PVN and SON the expressions of AVP and OT were more special than those in the parvocellular PVN. Oppositely, the expressions of CRH, TRH in the parvocellular were more special than the other two ; （3） The expressions of house keeping genes had no significant difference between labeled and non-labeled DMV neurons. Conclusion The quality and quantity of total RNA from unfixed brain tissues were better than fixed tissues applied in LMPC and the CTB tracer which may differentiate neurons had no significant effect on physiology of the neurons applied in LMPC. The results showed that the LMPC technique is suitable for the qualitative and quantitative study on individual neurons at mRNA level.
