Amplified RNA synthesized from limited quantities of heterogeneous cDNA.

TLDR

The heterogeneity of neural gene expression and the spatially limited expression of many low‑abundance messenger RNAs in the brain has made cloning and analysis of such messages difficult. To generate sufficient nucleic acids for standard cloning, we devised a method to produce amplified heterogeneous RNA populations from limited cDNA quantities. Whole cerebellar RNA was primed with a synthetic oligonucleotide bearing a T7 promoter, followed by second‑strand cDNA synthesis and T7 RNA polymerase‑mediated generation of amplified antisense RNA. We achieved up to 80‑fold molar amplification from nanogram cDNA, with amplified material matching parent size distribution and retaining sequence heterogeneity, enabling detection of moderate‑abundance mRNAs and single‑cell cDNA from cerebellar tissue.

Abstract

The heterogeneity of neural gene expression and the spatially limited expression of many low-abundance messenger RNAs in the brain has made cloning and analysis of such messages difficult. To generate amounts of nucleic acids sufficient for use in standard cloning strategies, we have devised a method for producing amplified heterogeneous populations of RNA from limited quantities of cDNA. Whole cerebellar RNA was primed with a synthetic oligonucleotide containing the T7 RNA polymerase promoter sequence 5' to a polythymidylate region. After second-strand cDNA synthesis, T7 RNA polymerase was used to generate amplified antisense RNA (aRNA). Up to 80-fold molar amplification has been achieved from nanogram quantities of cDNA. The amplified material is similar in size distribution to the parent cDNA and shows sequence heterogeneity as assessed by Southern and Northern blot analysis. Specific messages for moderate-abundance mRNAs for actin and guanine nucleotide-binding protein (G-protein) alpha subunits have been detected in the amplified material. By using in situ transcription to generate cDNA, sequences for cyclophilin have been detected in aRNA derived from single cerebellar tissue sections. cDNA derived from a single cerebellar Purkinje cell also has been amplified and yields material that hybridizes to cognate whole RNA and mRNA but not to Escherichia coli RNA.