RNA-seq

mRNA-seq, or mRNA-targeted RNA sequencing, is a next-generation sequencing (NGS) technique used to study the transcriptome - the set of mRNAs present in a given cell or tissue at a particular time. It provides a global view of gene expression, including quantitative and qualitative information about mRNA.

RNA-seq workflow

Applications of RNA-seq

Technical specifications of mRNA-seq (polyA)

• Library preparation: mRNA molecules with poly(A) tails are isolated from total RNA using magnetic beads coated with oligo(dT), which bind the poly(A) tails at the ends of mRNA. Total RNA is mixed with the beads, and after poly(A)-mRNA binding, the remaining RNA types (rRNA, tRNA) are removed by washing.
• Library preparation options:
  • Stranded - strand-specific library preparation. Strand orientation information is retained during sample preparation, making it possible to determine which DNA strand each RNA transcript originated from.
  • Non-stranded - library preparation without strand specificity. In this approach, RNA transcription direction is not retained, resulting in the loss of strand orientation information.
• Sequencing mode: 2x150 bp
• Sequencers: Illumina platform, NovaSeq 6000 and NovaSeq X sequencers.
• Recommended data output per sample:
  • 6 Gb per sample
  • 10 Gb per sample
  • 15 Gb per sample
  • Other configurations are also available depending on the client's needs.
• Data quality: Q30 ≥ 85%
• Recommended Sample Requirements - DNA/RNA:
  • Sample type: total RNA
  • Amount: > 0.5 μg
  • Concentration (NanoDrop): >50 ng/μL
  • Volume: >20 μL
  • Purity (OD260/280): 1.8 - 2.2
  • RIN: >7 (>6 for plants)
• MINIMUM Sample Requirements - DNA/RNA:
  • Sample type: total RNA
  • Amount: > 0.2 μg
  • Concentration (Qubit): >20 ng/μL
  • Volume: >20 μL
  • Purity (OD260/280): 1.7 - 2.5
  • RIN: >4.5 (>4 for plants)
  • 5.0 ≥ 28S/18S ≥ 1.0
• RNA isolation from the supplied samples is also available. Details here.

mRNA-seq (polyA) bioinformatics analysis

The advanced bioinformatics analysis for the mRNA-seq package includes, among other things:

• Data quality control
• Alignment to the reference genome
• Library quality control:
• SNP/InDel analysis
• Gene-structure optimisation
• Novel-gene analysis
• Gene-expression quantification
• Differential-expression analysis (DEG)
• Functional annotation of DEGs and enrichment analysis
• Differential alternative-splicing analysis
• Gene-set enrichment analysis (GSEA)
• Differential exon usage (DEU)

Example plots from the bioinformatics analysis report

Technical specifications of RNA-seq (rRNA depletion)

• Library preparation: depletion of ribosomal RNA, which can account for up to 90% of the sample's RNA, preserves the mRNA, lncRNA, circRNA and small-RNA fractions for analysis.
• Sequencing mode: 2x150 bp
• Sequencers: Illumina platform, NovaSeq 6000 and NovaSeq X sequencers.
• Recommended data output per sample:
  • 12 Gb per sample
  • 16 Gb per sample (the minimum data volume required to also analyse circRNA)
  • Other configurations are also available depending on the client's needs.
• Data quality: Q30 ≥ 85%
• Recommended Sample Requirements - DNA/RNA:
  • Sample type: total RNA
  • Amount: > 2000 ng
  • Concentration (NanoDrop): >100 ng/μL
  • Volume: >10 μL
  • Purity (OD260/280): 1.8 - 2.2
  • RIN: >7 (>6 for plants)
• MINIMUM Sample Requirements - DNA/RNA:
  • Sample type: total RNA
  • Amount: > 500 ng
  • Concentration (Qubit): >80 ng/μL
  • Volume: >10 μL
  • Purity (OD260/280): 1.7 - 2.5
  • RIN: >6.5 (>6 for plants)
  • 5.0 ≥ 28S/18S ≥ 1.0
• DNA extraction from submitted samples is also available. Details here.

RNA-seq (rRNA depletion) bioinformatics analysis

The advanced bioinformatics analysis for the RNA-seq package includes:

• Analysis of transcript types to identify mRNA and lncRNA.
• Study of relationships and dependencies between lncRNA and mRNA.
• Assessment of gene organisation, including intron and exon boundaries, alternative splicing and newly discovered genes.
• Measurement of lncRNA and mRNA expression levels in samples.
• Identification of genes (DEG) and lncRNAs (DE-lncRNA) differentially expressed between samples or groups.
• Assignment of functions to differentially expressed genes (DEG) and lncRNAs (DE-lncRNA), as well as identification of the biological pathways and processes in which they are involved.
• Identification of lncRNA-regulated target genes and analysis of their potential functions.
• Study of potential protein interactions associated with differentially expressed lncRNAs.
• Assessment of lncRNA sequence conservation across species to determine its potential biological significance.
• Review and classification of previously identified lncRNAs.
• Identification of potential miRNA precursors based on lncRNA sequences.
• Identification of miRNA-regulated target genes and study of their functions in biological processes.