A precise mRNA quantification method using CE-based SSCP.
Even though mRNA quantification provides significant information for biological analysis, current methods such as Northern blot analysis and real-time PCR are known to be laborious and lacking in precision. In this study, we demonstrate a new precise mRNA quantification method using CE based on SSCP (CE-SSCP) coupled with reverse transcription. mRNA samples could be simply analyzed for the quantification directly with reverse transcript obtained from a single reaction. This helps to avoid considerable errors generated by a series of the tedious manual steps. Also, unlike real-time PCR, reverse transcripts can be directly quantified by CE-SSCP in this method without further data estimation. Reproducibility and accuracy of CE-SSCP for mRNA quantification was examined using enhanced green fluorescent protein (eGFP) mRNA transcribed in vitro. Specific reverse transcription primer was determined for the accurate quantification of eGFP mRNA from total RNA obtained from the recombinant Escherichia coli. Using elongation factor Tu mRNA as an internal standard, it was shown that sample-to-sample variation could be minimized. Expression kinetics at both mRNA level and protein level was studied and the potential of CE-SSCP in expression analysis was demonstrated by comparison with the eGFP activity assay.
Park
Young Seoub
YS
Department of Chemical Engineering, POSTECH, Hyoja-dong, Pohang, Gyeongbuk, Korea.
Chu
Hun Su
HS
Hwang
Seung Ha
SH
Seo
Jong Hyuk
JH
Choi
Cha Yong
CY
Jung
Gyoo Yeol
GY
D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007700:Kinetics; D018807:Polymorphism, Single-Stranded Conformational; D012333:RNA, Messenger / Q000032:analysis*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D012333:RNA, Messenger; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins