22] Receptor mRNA measurement by multiplex nuclease protection assay

Abstract

This chapter discusses receptor mRNA measurement by multiplex nuclease protection assay. The methods most commonly employed in mRNA analysis can be divided into four groups: (i) filter hybridization techniques, such as northern blot and dot blot; (ii) solution hybridization–nuclease protection; (iii) reverse transcriptase–polymerase chain reaction (RT–PCR); and (iv) in situ hybridization histochemistry. These techniques differ in a number of factors such as type of probe used, method of signal detection, type and amount of information obtained, specificity, background, sensitivity, quantitative accuracy, and sample handling. The types of probe most commonly used in hybridization techniques include DNA, RNA, and deoxyoligonucleotide probes. DNA and RNA probes, usually 100–1000 bases in length, are typically generated using a labeled nucleotide that is incorporated uniformly along the sequence. With deoxyoligonucleotide probes (usually 20–50 bases in length) the labeled nucleotide is added at the end of the molecule. Double-stranded DNA probes are commonly used in dot blot and northern blot analyses.