Nuclear and cytoplasmic mRNA quantification by SYBR green based real-time RT-PCR.
Measurement of the steady-state abundance of nuclear and cytoplasmic RNA requires efficient subcellular fractionation and RNA recovery coupled with accurate quantification of individual RNA species. Detergent lysis of tissue culture cells provides a simple fractionation procedure that can be optimized to individual cell lines. The large dynamic range, extreme sensitivity, high sequence-specificity, and fast turn-around time has allowed real-time reverse transcription polymerase chain reaction (real-time RT-PCR) to become a standard tool for mRNA quantification. Among the different chemistries used for PCR product detection during amplification, DNA binding dyes such as SYBR Green I are simple, versatile, and yet highly reliable and least expensive. With attention to primer design and cycling conditions, virtually any mRNA species can be accurately quantified from even minute quantities of starting RNA. This method provides an accurate and efficient procedure for estimating the relative ratios of nuclear and cytoplasmic RNA concentrations.
Wang
Yaming
Y
Department of Pathology, NYU Cancer Institute, New York University School of Medicine, 550 1st Avenue, New York, NY 10016, USA.
Zhu
Wei
W
Levy
David E
DE
D052160:Benzothiazoles; D002467:Cell Nucleus / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry*; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism*; D009930:Organic Chemicals / Q000378:metabolism*; D011804:Quinolines; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; C098022:SYBR Green I