Application of laser-induced fluorescence for quantitative analysis of messenger RNA

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Cloning of casein mRNA and competitive reverse-transcription/polymerase chain reaction (RT-PCR) mRAN was extracted from lactating mouse mammary gland. Among casein mRNAs, mRNA for casein with the molecular weight of 22,000 was transcribed to cDNA and cloned. The nucleic acid sequence of casein cDNA was determined. DNA fragment of known foreign gene was inserted into casein cDNA.This kimeric DNA was used as competitor DNA during the polymerase chain reaction.Animals and assay conditions The quantity of casein mRNA in the mammary gland was determined by the RT-PCR throughout lactation. Its quantity was small at early lactation, reached maximum at midlactation, and declined gradually toward to the end of lactation. By the removal of pups from the midlactating mother, casein mRNA began to disappear from the mammary gland at 9h after the separation. By supplying the foser pups, casein mRNA increased and returned to the normal level.Fluorescene-labelled probe First, casein cDNA was amplified with fluorescene-labelled UTP by the RT-PCR.The incorporation efficiency was very poor. Secondly, the casein DNA fragment of 400 bp in lenght was inserted into the plasmid DNA.After the digestion of the DNA with Spe I,the fluorescene-labelled sense and antisense RNA probes were synthesized using T7 and SP6 polymerases, respectively. We detected the fluorescene-labelled RNA probe with the amount of larger than 10^<-15> mol and could be used for tne Northern blot analysis and for capillary clectrophoresis.