In situ localization of PCR-amplified human and viral cDNAs.

Abstract

We describe a technique, called reverse transcriptase (RT) in situ PCR, whereby RNA may be nonisotopically detected in fixed cells when amplified by PCR after cDNA synthesis by RT. RT in situ PCR using primers specific for the measles virus generated an intense signal in most measles-infected HeLa cells, as compared to the weak signal generated in few cells using standard in situ hybridization analysis. The viral RNA that localized to the nucleus spared the nucleoli, was most evident when the RT step used the primer complementary to the negative genomic strand, and was demonstrated in all multinucleated cells and the majority of uninucleate cells. A hybridization signal was evident with standard RNA in situ hybridization using the human megakaryocyte cell line Dami and a probe for glycoprotein liB (GLIB) mRNA but not a probe for amyloid precursor protein (APP) or gelsolin (GEL) mRNA. After RT in situ PCR, signals were evident for each target localizing to the nucleolus for APP and to perinucleolar and cytoplasmic locations for GEL and GLIB. The latter findings suggest that mRNAs may follow different geographic pathways as they progress from premessage to transcriptionally active message.The study of the cellular and tissue distribution patterns of DNA and RNA has been greatly facilitated by in situ hybridization. However, one limitation of in situ hybridization is its relatively high detection threshold, which is about 20 copies per cell. (1~ Recent work on the processing of RNAs has focused on the nuclear matrix, which appears to provide a specific and obligatory pathway for this processing from transcription to splicing up to and including release of translationally active message into the cytoplasm. (z-6) One observation supporting this hypothesis is the intimate association of the specific small nuclear (sn) RNAs and their associated proteins in spliceosomes, which are essential for splicing of pre-mRNA, with the nuclear matrix. (7,8) The inability to detect low copy numbers of RNA has hindered the study of the dynamics and subcellular organization of mRNA transcription, processing, and transport. Wang et al. (9) injected labeled 13-globin pre-mRNA into nuclei and observed discrete subnuclear compartmentalization of the signal. Others have noted a dramatic compartmentalization of pre-mRNAs in the nucleus. (1~ Detection of in situ PCR-amplified DNA from a single DNA molecule per cell has been described, (~'~1~ and the 100-fold amplification is readily detected by in situ hybridization analysis. The hot start modification to the PCR in situ technique was used to improve sensitivity by increasing the amount of specific target synthesis and reducing the nonspecific amplification that may follow extension of primers annealed to nontarget sequences (mispriming) and extension of primers after they anneal over parts of their sequence to form primer oligomers. This enhanced specificity allows the incorporation of a labeled nucleotide into the amplified product with the assurance that the resultant signal reflects tar...

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