[39] Measurement of apolipoprotein mRNA by DNA-excess solution hybridization with single-stranded probes

Nucleic acid hybridization provides an invaluable method for the detection and measurement of specific mRNA molecules, and includes basically three hybridization methods: RNA-excess solution hybridization or R0t analysis, hybridization to immobilized RNA or blot hybridizations, and DNA-excess solution hybridization. This chapter describes the hybridization assay and two methods for the preparation of high specific activity single-stranded probes. Blot hybridization is usually carried out with RNAs that have been resolved by gel electrophoresis and transferred to nitrocellulose paper (Northern analysis) or after direct application to the paper (dot–blot analysis). The dot–blot procedure is simple, requires a minimum of probe preparation, and is a quantitative method. However, DNA-excess solution hybridization provides the greatest sensitivity and reliability for mRNA measurement. This method has several advantages including the fact that the assay is carried out in solution such that all the mRNA and probe are available to react. This method provides absolute values for mRNA measurements, and the method is absolutely sensitive.