The successes and future prospects of the linear antisense RNA amplification methodology

Abstract

It has been over a quarter of a century since the introduction of the linear RNA amplification methodology known as antisense RNA (aRNA) amplification. Whereas most molecular biology techniques are rapidly replaced owing to the fast-moving nature of development in the field, the aRNA procedure has become a base that can be built upon through varied uses of the technology. The technique was originally developed to assess RNA populations from small amounts of starting material, including single cells, but over time its use has evolved to include the detection of various cellular entities such as proteins, RNA-binding-protein-associated cargoes, and genomic DNA. In this Perspective we detail the linear aRNA amplification procedure and its use in assessing various components of a cell's chemical phenotype. This procedure is particularly useful in efforts to multiplex the simultaneous detection of various cellular processes. These efforts are necessary to identify the quantitative chemical phenotype of cells that underlies cellular function.

Figure 1. Schematic linear antisense RNA (aRNA) amplification procedure.

In the first round, first-strand cDNA synthesis by reverse transcription (RT) is primed from mRNA after oligo(dT)-T7 primer anneals to the poly(A) tail of mRNA. RNase H is then used to digest portions of the bound mRNA to create RNA fragments that serve to prime second-strand cDNA by DNA polymerase (pol). Finally, aRNA is amplified via linear in vitro transcription by T7 RNA polymerase, using the T7 RNA polymerase promoter incorporated in the double-stranded cDNA. In the second round, first-strand synthesis is primed by random primers instead of the oligo(dT)-T7 primer by reverse transcriptase using the aRNA as a template instead of mRNA. After RNA denaturation, second-strand synthesis is primed with the oligo(dT)-T7 primer, which binds to the poly(A) tail of the cDNA created during first-strand synthesis by DNA polymerase. Finally, RNA is again linearly amplified through the enzymatic activity of T7 RNA polymerase acting on its promoter that is incorporated into the double-stranded cDNA2,3. Credit: Marina Spence/Springer Nature.

Figure 2. Schematic overview of applications of linear aRNA amplification for the detection of biological chemicals.

(a) Transcriptome detection (RNA-seq). After two rounds of linear aRNA amplification from isolated single cells, an Illumina TruSeq library is generated as outlined. Because of the short length of the aRNA amplified by the procedure, the step that breaks long RNA into smaller parts in the original Illumina library protocol can be omitted. For strand specificity, deoxyuridine triphosphate is incorporated into the second-strand cDNA. After sequencing adaptor ligation, the cDNA fragments with adaptors at both end are PCR amplified, and ready for sequencing7,8. (b) RNA-binding protein (RBP)/RNA cargo detection (APRA). An antibody (Ab) to RBP is conjugated to an oligonucleotide and applied to fixed cells. After antibody–RBP binding, the oligonucleotide (containing the T7 promoter sequence) is positioned closely enough to the RNA to prime first-strand cDNA synthesis in situ. After second-strand synthesis in vitro, the antibody is removed by restriction enzyme cleavage and the aRNA is linearly amplified by in vitro transcription using the T7 promoter incorporated in the cDNA. The aRNA product is suitable for PCR, microarray, and next-generation sequencing analysis43. (c) Protein detection (IDAT). A detection antibody is first generated by conjugation of a target-protein-specific antibody to a double-stranded (ds) oligonucleotide containing T7 promoter. In a 96-well plate, a capture antibody then binds to the antigen (Ag) of interest from a sample. After the addition of detection antibody to the sample, RNA is linearly amplified by T7 RNA polymerase from the double-stranded oligonucleotide template incorporating the T7 promoter. The amount of RNA product is indicative of the original amount of antigen in the sample and can be used for fluorimeter detection, PCR. or sequencing45. (d) Whole-genome DNA amplification (LIANTI). A LIANTI transposon is first created by joining of the T7 promoter site to a transposase-binding site. This transposon is then mixed with transposase to generate the LIANTI transposome. After the LIANTI transposome is mixed with DNA isolated from single cells, the transposase mediates random insertion of LIANTI transposon into the DNA and subsequent excision of genomic DNA, which is followed by DNA polymerase gap extension. After the addition of T7 RNA polymerase, single-stranded aRNA is generated that is capable of self-priming on the 3Ś end. After reverse transcription, RNase treatment, and second-strand synthesis, double-stranded LIANTI amplicons tagged with unique molecular barcodes are formed and ready for DNA library preparation and next-generation sequencing11. Pol, polymerase; IVT, in vitro transcription. Credit: Marina Spence/Springer Nature

Figure 3. A timeline of selected biological uses of linear aRNA amplification.

The applications of linear aRNA amplification have steadily increased since its original publication in 19902. There was a burst of technique development and applications during the five years after its initial publication, and a second burst of technique modifications and uses after 2010, when next-generation sequencing was introduced. This timeline presents selected highlights and is not exhaustive, as over this period thousands of papers were published on the use of linear aRNA amplification in different experiments (representative reference citations are shown in parentheses). Credit: Marina Spence/Springer Nature.