Overview - What is RNA-seq?
The transcriptome is a collection of all RNA present in a cell or a population of cells at any given moment. The transcriptome is dynamic, as the levels of RNA transcripts vary during different developmental stages or in response to certain conditions.
Transcriptome sequencing or RNA-seq is a next-generation sequencing based approach to profiling and analysing RNA. This technique delivers unbiased information without the need for prior knowledge of the genome or transcriptome. Transcriptome sequencing is often the method of choice for analysis of differentially expressed genes, as well as for RNA editing and profiling of allele-specific gene expression. RNA-seq can also be used to investigate splicing patterns, splicing variants, gene isoforms, single nucleotide polymorphisms and post transcriptional modifications.
Applications - What are the advantages of RNA-Seq?
Sequencing of RNA transcripts is a beneficial method for RNA profiling because it allows for:
- Digital gene expression (DGE) analysis – sensitive quantification of gene expression levels and transcriptional activity
- Identification of both known and novel transcripts
- Analysis across a wide dynamic range – detection of more differentially expressed genes with higher fold change
- Profile the RNA transcripts of nearly any organism without the need for a reference sequence
- Complete view of the entire transcriptome
Workflow - Methods & technologies for RNA-seq
Transcriptome sequencing begins with isolating RNA and converting this RNA into so-called complementary DNA (cDNA). Total RNA or RNA types like mRNA or small RNA can be investigated. Strand-specific or random-primed libraries can be created based on how the cDNA is synthesised. Once adapters are ligated to the cDNA fragments, the cDNA undergoes single-read or paired-end sequencing. The sequencing reads are either assembled de novo or they are aligned to a reference genome or transcriptome. The reads can be quantified to evaluate differential gene expression levels or analysed further to provide data on transcript variants and posttranscriptional modifications.
Microarray vs. RNA-seq
RNA-seq is an evolving technology, whereas microarrays are the traditional method for differential gene expression analysis. RNA-seq offers distinct advantages over hybridisation-based methods. Next-generation sequencing of transcriptomes does not rely on pre-designed primers and probes, which eliminates content bias and provides a truly genome-wide view of the transcriptome. RNA-seq provides the possibility of exploring coding and non-coding RNA with more sensitivity, higher dynamic range and with single-base resolution. NGS-based sequencing of cDNA can reveal complex regions like exon-exon junctions or sequence variations that cannot be profiled by hybridisation methods. Multiplexing capabilities and the ability to store and re-analyse data without repeated experiments render NGS of transcriptomes more cost-effective than microarrays.
Scientific expertise: RNA-seq
Eurofins Genomics is an experienced provider of RNA-seq services. With several specialised sequencing products to serve the transcriptomics field, Eurofins Genomics helps answer questions regarding gene function on a daily basis. Eurofins Genomics uses Illumina’s or PacBio’s sequencing technologies, for flexible sequencing coverage. The RNA-seq services can help further various research objectives, from screening to discovery projects, as well as de novo transcriptome analysis and isoform sequencing (Iso-seq)-driven research.
Transcriptome analysis at Eurofins Genomics has helped elucidate cardiac remodelling mechanisms, post-transcriptional gene silencing, features of CD4+ T cells in coeliac disease, characteristics of photosynthetic genes in maize, the molecular diversity of snake venom, strategies for obtaining human-induced pluripotent stem-cell-derived neurons and many others.
Products related to cDNA sequencing
Are you planning a project that needs transcript sequencing? Eurofins Genomics offers whole-transcriptome sequencing, mRNA sequencing and small RNA sequencing services to provide in-depth analysis of differential gene activity, alternative splicing and expression of gene isoforms. Our services combine RNA isolation, rRNA depletion, library preparation, sequencing on state-of-the-art Illumina platforms, and expert transcriptomic data analysis.
Contact our Customer Care for more information.
Did you know that Eurofins Genomics also has unrivalled expertise in Sanger Sequencing that converges into a comprehensive product Portfolio?