RNA analysis would do well to follow the example set by naturalists, scientists who patiently add new species to their catalogs while remembering that any catalog, however comprehensive, is of limited interest. What really matters, in botany or zoology or, more pertinently, RNA biology, is learning how species impact each other and fulfill their
Even RNA biology’s most familiar species, such as messenger RNA (mRNA), are less domesticated than one might suppose. And relatively unfamiliar species—such as microRNA (miRNA), small interfering RNA (siRNA), circular RNA (circRNA), long noncoding RNA (lncRNA), small nucleolar RNA (snoRNA), and extracellular RNA (exRNA)—are bound to exhibit interesting behaviors whether they are in the wild or, should field work be impractical, the laboratory.
RNA analysis has contributed to the identification of many new classes of small and large noncoding RNAs. Moreover, it has shown that noncoding RNAs fulfill novel catalytic and transcriptional regulatory functions. Finally, it has started to uncover the functional significance of the chemical modifications that RNAs may undergo.
RNA analysis continues to evolve. It is improving its ability to cope with small, low-quality samples; to disregard inconveniently abundant transcripts; and to generate quantitative and structurally informative transcriptome profiles. It is also beginning to integrate transcriptomics with other “omics,” such as proteomics, metabolomics, and epigenomics.
Working with challenging samples
If the quantity of material to be processed for RNA analysis is minimal, RNA analysis may need to incorporate technology such as deep sequencing, says G. Brett Robb, PhD, scientific director, RNA and genome editing, New England Biolabs (NEB). According to Robb, the NEBNext Ultra II RNA Library Prep kits allow the use of lower input amounts, as small as 5 ng, and require fewer PCR cycles. Also, the kits allow investigators to increase the complexity and transcript coverage of their libraries. Another kit, the NEBNext Single Cell/Low Input RNA Library Prep kit for Illumina, facilitates single-cell analysis, utilizing as little as 2 pg–200 ng total RNA per cell.
Another demand is the depletion of ribosomal RNA (rRNA) or abundant transcripts from total RNA samples. rRNAs constitute 80–90% of total RNA. Efficient removal is critical for cost-effective sequencing of RNA samples. Depletion can be especially challenging if investigators must work with low-input amounts and low-quality RNA, such as RNA extracted from formalin-fixed paraffin-embedded (FFPE) samples. The NEBNext rRNA Depletion kits for human, mouse, and rat samples employ the RNase H method, as well as complete probe tiling of rRNA, are designed to ensure that even degraded rRNA is hybridized and subsequently removed.
Aiming to facilitate overall ease of use, NEB has developed WarmStart technology, which can allow room temperature set up of RT-qPCR experiments. The company asserts that WarmStart is particularly well suited for use with detection methods that rely on loop-mediated isothermal amplification (LAMP). Reverse transcriptases are coupled with reversibly bound aptamers that inhibit activity at room temperature.
As the reaction heats up, the enzyme
becomes fully active.
Characterizing RNA modifications
“A fascinating new direction in RNA biology is characterizing the chemical modifications of RNA—the epitranscriptome—and understanding the functional consequences of epitranscriptomic marks,” discusses Robb. “Being able to read and write these modifications site-specifically will be increasingly important.
“The development of RNA modification detection enzymes and workflows as well as novel RNA enzymes, such as ligases and end-modifying enzymes, will broadly enable analysis of more well-known epitranscriptomic marks, giving researchers new tools to create new analysis workflows. For RNA therapeutics, the thorough characterization of RNA molecules will be critical.”
N6-methyladenosine, otherwise known as m6A, is the most abundant internal modification in mRNA and plays an important biological function in the regulation of different cellular processes. The Epimark m6A enrichment kit uses a highly specific antibody to m6A to immunoprecipitate m6A RNA. The enriched m6A-containing RNA can then be analyzed by RT-qPCR or deep sequencing.
A cocktail of enzymes, a Nucleoside Digestion Mix, can be used to reduce RNA or DNA to component nucleosides that can then be analyzed by LC–MS for quantitation and discovery of new RNA modifications and epitranscriptomic marks.
Making functional RNA molecules by in vitro transcription, including mRNAs is another key RNA technique, adds Robb. mRNA can be easily chemically transfected or electroporated into cell models to assess gene function in a DNA-independent way, an approach that is particularly powerful when coupled with CRISPR-Cas gene inactivation or siRNA knockdown. The HiScribe line of in vitro transcription kits is designed to permit microgram to milligram–scale synthesis of mRNA or RNA and work with Monarch RNA purification columns for facile synthesis and purification of RNA and mRNA molecules.
Microarrays and RNA-seq
“RNA has always been a challenging molecule due to its limited stability,” cautions Raymond Miller, PhD, global product manager, Diagnostic and Genomic Group, Agilent Technologies. “Despite many improvements, there are still several significant issues to various RNA analysis approaches.
“For example, increasingly, basic researchers, translational researchers, and clinicians are seeking to obtain discovery and validation RNA analysis data from difficult sample types. These challenging samples include FFPE, single-cell, and ‘circulating’ or exosomal RNA, and are often characterized by being of low quantity and quality.”
Researchers have shown that they can successfully run both RNA sequencing (RNA-seq) and microarray experiments with samples previously considered unusable. Accordingly, Agilent has focused efforts on developing chemistries and dedicated protocols that work optimally with small amounts of RNA and better tolerate the inhibitory effects of sample matrices.
The SureSelect RNA target enrichment and microarray platform is a core example. Probes have been developed that enrich relevant RNA targets with improved efficiency, while minimizing enrichment bias.
“Our R&D team developed an miRNA microarray solution that includes detection probes that are designed to target only mature miRNA, and a labeling solution that directly labels miRNA molecules, limiting bias introduced,” notes Miller. “We have many ongoing projects to develop new targeted RNA-seq workflows based on newer chemistries, target-enrichment algorithms, microarray customization, and data analysis capabilities.”
The RNA analysis market is rapidly growing, along with a need to technically and functionally validate clinically relevant RNA, and an increase in the development of RNA-based laboratory developed tests (LDTs). Agilent projects that both researchers and clinicians will embrace multiomics approaches that utilize RNA in conjunction with other analytes, such as DNA and methylated-DNA. Further refinement of microarrays and next-generation sequencing (NGS) technologies will occur with a growing emphasis on developing fully integrated sample-to-insight workflows, along with products to address an increased need for more customization to enrich specific RNA.
NGS has revolutionized many areas of genomics research including the study of the transcriptome. RNA-seq is a highly sensitive and accurate method of measuring expression across the transcriptome. It provides visibility to previously undetected changes occurring in various disease states, in response to therapeutics, under different environmental conditions, and across a broad range of other study designs without the limitation of prior knowledge.
RNA-seq lets researchers find both known and novel features in a single
assay, permitting the detection of transcript isoforms, gene fusions, single nucleotide variants, allele-specific gene expression, and other features.
Coping with sample diversity
“In general, the biggest challenge for RNA-seq is the diversity of the sample itself,” states Gary Schroth, PhD, distinguished scientist and vice president, Illumina. “Quality and processing can be highly variable as well as the amount of RNA. Our kits are designed to handle a wide range of sample input from as low as 1 ng.
“RNA-seq with NGS is increasingly the method of choice for researchers studying the transcriptome. It offers a broader dynamic range enabling more sensitive and accurate measurement of gene expression and can be applied to any species, even if reference sequencing is not available.”
Schroth adds that push-button RNA-seq software tools may be packaged in an intuitive user interface especially designed for biologists: “These user-friendly tools support a broad range of NGS studies, from gene expression analysis to total RNA expression profiling. Most recently, sets of unique dual indexes have been released that can be used not only for RNA but also for other nucleic acid types, allowing for flexibility and scalability.”
In the fall of 2018, Illumina announced the launch of TruSight Oncology 500 (TSO 500), a comprehensive RUO pancancer assay designed to identify known and emerging tumor biomarkers. TSO 500 utilizes both DNA and RNA from subject tumor samples to identify key somatic variants underlying tumor progression, such as small DNA variants, fusions, and splice variants. It can also measure tumor mutational burden and microsatellite instability, features that are potentially key biomarkers for emerging immunotherapies.
In addition, a single-cell analysis application with the SureCell WTA 3′ Library Prep kit was developed in collaboration with Bio-Rad and introduced with Bio-Rad’s ddSEQ instrument.
RNA analysis services
Novogene provides quantitative and structural analysis services for various types of RNA, including mRNA, small RNA, lncRNA, and circRNA, along with more comprehensive epigenomic analysis services such as whole-genome bisulfite sequencing (WGBS), chromatin immunoprecipitation followed by sequencing (ChIP-seq), and assay for transposase accessible chromatin sequencing (ATAC-seq). ATAC-seq is a fast and sensitive alternative to DNase-seq for assaying chromatin accessibility genome-wide, or to MNase for assaying nucleosome positions in accessible regions of the genome.
“Microfluidic-based single-cell transcriptome technology has attracted extensive interest from scientists worldwide. We use a combination of multiple complementary platforms, including 10x Genomics’ Chromium and BD’s Rhapsody, to perform single cell transcriptomics,” states Jun Wu, vice president, global NGS service, Novogene.
Synergistic analysis of multiple types of RNA and integrated omics analysis combining genomics, epigenomics, proteomics, and metabolomics is another hurdle for the field. Novogene is building comprehensive multiomics experiment and analysis systems that incorporate NGS platforms (including platforms from Pacific Biosciences and Oxford Nanopore Technologies) and high-resolution mass spectrometers to solve complex research issues. Flexible cloud computing is used to speed up and economize RNA analysis.
“With the foreseeable breakthroughs in PacBio and Nanopore sequencing technologies, we expect to see greater popularity of full-length transcriptome sequencing for comprehensive quantitative and structural analysis,” Wu declares. “In addition, the vigorous development of many epigenomic technologies, including WGBS, RNA methylation, and ATAC-seq, are deepening our understanding of RNA analysis. We will continue to collaborate closely with upstream technology developers and scientists to lead these technological trends.”