Objective Detection and quantification of RNA synthesis in cells is a widely used technique for monitoring cell viability,health,and metabolic rate.After exposure to environmental stimuli,both the internal reference g...Objective Detection and quantification of RNA synthesis in cells is a widely used technique for monitoring cell viability,health,and metabolic rate.After exposure to environmental stimuli,both the internal reference gene and target gene would be degraded.As a result,it is imperative to consider the accurate capture of nascent RNA and the detection of transcriptional levels of RNA following environmental stimulation.This study aims to create a Click Chemistry method that utilizes its property to capture nascent RNA from total RNA that was stimulated by the environment.Methods The new RNA was labeled with 5-ethyluridine(5-EU)instead of uracil,and the azido-biotin medium ligand was connected to the magnetic sphere using a combination of“Click Chemistry”and magnetic bead screening.Then the new RNA was captured and the transcription rate of 16S rRNA was detected by fluorescence molecular beacon(M.B.)and quantitative reverse transcription PCR(qRT-PCR).Results The bacterial nascent RNA captured by“Click Chemistry”screening can be used as a reverse transcription template to form cDNA.Combined with the fluorescent molecular beacon M.B.1,the synthesis rate of rRNA at 37℃is 1.2 times higher than that at 15℃.The 16S rRNA gene and cspI gene can be detected by fluorescent quantitative PCR,it was found that the measured relative gene expression changes were significantly enhanced at 25℃and 16℃when analyzed with nascent RNA rather than total RNA,enabling accurate detection of RNA transcription rates.Conclusion Compared to other article reported experimental methods that utilize screening magnetic columns,the technical scheme employed in this study is more suitable for bacteria,and the operation steps are simple and easy to implement,making it an effective RNA capture method for researchers.展开更多
玻璃体化冷冻胚眙在国际上早有报道,但能够受孕、生出牛犊的报道不多,在中国只是首例。对小鼠和家兔的胚胎在实验室已经常规化的应用玻璃体化冷冻技术。近年来自1986年Scheffen等用10％甘油和25％1,2丙二醇作为防冻剂冷冻鼠胚,Massip et...玻璃体化冷冻胚眙在国际上早有报道,但能够受孕、生出牛犊的报道不多,在中国只是首例。对小鼠和家兔的胚胎在实验室已经常规化的应用玻璃体化冷冻技术。近年来自1986年Scheffen等用10％甘油和25％1,2丙二醇作为防冻剂冷冻鼠胚,Massip et al(1986)冷冻牛胚之后,我们对牛胚胎的玻璃体化冷冻进行了部分研究。 本研究应用三种不同种和不同浓度的保护剂处理7天的桑椹胚和囊胚采用玻璃体化冷冰法进行冷冻。在液氮中保存一段时间后,解冻脱掉保护剂,移植给受体母牛,先后有3头受体牛妊娠,目前产犊1头。展开更多
文摘Objective Detection and quantification of RNA synthesis in cells is a widely used technique for monitoring cell viability,health,and metabolic rate.After exposure to environmental stimuli,both the internal reference gene and target gene would be degraded.As a result,it is imperative to consider the accurate capture of nascent RNA and the detection of transcriptional levels of RNA following environmental stimulation.This study aims to create a Click Chemistry method that utilizes its property to capture nascent RNA from total RNA that was stimulated by the environment.Methods The new RNA was labeled with 5-ethyluridine(5-EU)instead of uracil,and the azido-biotin medium ligand was connected to the magnetic sphere using a combination of“Click Chemistry”and magnetic bead screening.Then the new RNA was captured and the transcription rate of 16S rRNA was detected by fluorescence molecular beacon(M.B.)and quantitative reverse transcription PCR(qRT-PCR).Results The bacterial nascent RNA captured by“Click Chemistry”screening can be used as a reverse transcription template to form cDNA.Combined with the fluorescent molecular beacon M.B.1,the synthesis rate of rRNA at 37℃is 1.2 times higher than that at 15℃.The 16S rRNA gene and cspI gene can be detected by fluorescent quantitative PCR,it was found that the measured relative gene expression changes were significantly enhanced at 25℃and 16℃when analyzed with nascent RNA rather than total RNA,enabling accurate detection of RNA transcription rates.Conclusion Compared to other article reported experimental methods that utilize screening magnetic columns,the technical scheme employed in this study is more suitable for bacteria,and the operation steps are simple and easy to implement,making it an effective RNA capture method for researchers.
文摘玻璃体化冷冻胚眙在国际上早有报道,但能够受孕、生出牛犊的报道不多,在中国只是首例。对小鼠和家兔的胚胎在实验室已经常规化的应用玻璃体化冷冻技术。近年来自1986年Scheffen等用10％甘油和25％1,2丙二醇作为防冻剂冷冻鼠胚,Massip et al(1986)冷冻牛胚之后,我们对牛胚胎的玻璃体化冷冻进行了部分研究。 本研究应用三种不同种和不同浓度的保护剂处理7天的桑椹胚和囊胚采用玻璃体化冷冰法进行冷冻。在液氮中保存一段时间后,解冻脱掉保护剂,移植给受体母牛,先后有3头受体牛妊娠,目前产犊1头。
