Calibration-free quantitative analysis of mRNA.
Here we introduce a method for accurate and sensitive quantitative analysis of mRNA, which does not require calibration with mRNA. The method uses a fluorescently labeled hybridization probe as a reference standard. It involves the following: (i) annealing mRNA to the excess of the fluorescently labeled ssDNA hybridization probe, (ii) separation of the mRNA-probe hybrid from the excess of the probe by gel-free capillary electrophoresis mediated by ssDNA-binding protein, (iii) fluorescence detection of the hybrid and the excess probe, and (iv) quantification of mRNA using a simple algebraic formula. The method also overcomes a number of other limitations of conventional methods: the entire procedure currently takes only 2 h and accurately quantifies 10(5) copies of mRNA. With further improvements to the method, the procedure can be potentially shortened to 10 min, and the limit of quantification can be decreased to as few as 100 copies of mRNA. In this work, we prove the principle of the method by quantifying mRNA of green fluorescent protein in the matrix of total cellular RNA. The developed method is quantitative, simple, fast, and highly sensitive. It requires commercially available instrumentation only. The method will be an indispensable tool for molecular and cell biology studies.
Al-Mahrouki
Azza A
AA
Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada.
Krylov
Sergey N
SN
D019075:Electrophoresis, Capillary; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000235:genetics; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis*; D012015:Reference Standards; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins