Quantification of mRNA Levels Using Ribonuclease Protection Assay

互联网2014-02-14

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Several methods are available for the quantification of neurotrophin mRNA levels. This chapter describes the RNAse protection assay (RPA) first publishcd by Zinn and co-workers ( 1 ) and developed further by Melton and co-workers ( 2 ). In this method, a radioactively labeled antisense RNA probe is hybridized in solution with a purified RNA sample. The labeled probe forms double-stranded RNA-RNA hybrids with complementary mRNA molecules. Separation of RNA-RNA hybrids from free probe and sample RNA is achieved by digestion with RNAses that specifically degrade single-stranded RNA. After these RNAses have been inactivated by proteinase K digestion, protected RNA fragments are separated in ultrathin acrylamide gels and visualized with autoradiography. The labeled antisense RNA probe is derived from a plasmid construct that has a bacterial RNA polymerase promoter such as SP6, T3, or T7 downstream of a sequence that encodes a portion of neurotrophin mRNA. This DNA fragment can either be part of a cDNA clone or a genomic subfragment, but it should be selected so that the plasmid construct can be digested with a specific restriction enzyme prior to transcription, to yield a labeled RNA species from 100 to 400 nucleotides long ( see Fig. 1 ). As RNAses can detect mismatches of one basepair, the probe has to be derived from the same species as the RNA being studied, or if it is heterologous, it has to correspond to a region where its nucleotide sequence is fully conserved between the two species.

Fig. 1. Generation of antisense RNA-probe and synthetic mRNA from plasmid construct. ( A ) An internal BglII-SacII fragment of the human NT-3 cDNA was cloned into the pBluescript vector between the T3 and T7 RNA-polymerase promoters. Note that the two promoters are oriented so that opposite DNA strands will be transcribed into RNA. ( B ) To synthesize the antisense probe, the plasmid is cut with Hind III within the polylinker. Transcription of this linearized plasmid with T3 RNA polymerase will result in the noncoding strand being synthesized. The antisense probe has some vector-derived sequences at both 5′and 3′ends (shown by the gray color). ( C ) To prepare synthetic NT-3 mRNA, the plasmid is cut with PvuII , which cuts twice in the plasmid backbone. The presence of the vector-derived PvuII fragment will not interfere with transcription with T7, as it has no promoter sites. The coding mRNA strand is synthesized by T7 RNA-polymerase. The synthetic mRNA will have some vector-derived sequences at both ends (shown by the gray color).