Comprehensive view of transcription profiles

Standard gene-expression analysis is performed on protein-coding RNA, since this provides you the fastest and affordable method to visualize gene-expression. However, with Total Transcriptome, you extend your vision towards all transcripts, including long non-coding RNA (lncRNA) and cicular RNA (circRNA) species.

Total RNA sequencing helps you to overcome common causes for RNA degradation, such as FFPE-fixation and laser-capture methods. Additionally, precious samples from a cohort might not be handled optimally, blocking the only source to obtain transcriptome profiles for your study. Our scientists have set up validated procedures to analyze these challenging samples. This includes clinical-grade sample isolation from FFPE material and tailored data-analysis.

A ribosomal RNA (rRNA) depletion step is usually required, since the 16S and 18S ribosomal subunits make up ~60-90% of all RNA species present in your sample. You make better use of the sequencing space by removing them at the sample preparation phase. What remains is messenger RNA, and many other regulatory types of RNA.

Working seamlessly with you on your project:

• Measurement of all RNA transcripts present
• rRNA depletion and globin reduction are optional
• Challenging samples: The average RNA transcript size must exceed 50 bp to allow for unique mapping and analysis. Check your (column-)isolation procedure to determine from which size (bp) the RNA fragments are retained.

• Optimal total RNA input: 100 ng / sample
• Ideally deliver ≥250 ng RNA to allow for QC and normalization
• Minimally validated RNA input: 10 ng
• For RIN / RQN of all qualities

• Stranded sequencing of all RNA transcripts, including rRNA depletion
• Quality score Q30 of ≥80% for PE 150 reads
• TAT: 3 weeks

• Sequencing on NovaSeq 6000 PE150
• Gene-expression analysis: 40 million reads

The specifications of the data-set that you will recieve are listed in your personal quotation. Generally, the sequencing quality score is Q30≥80% for PE 150 reads (Illumina’s official guarantee ≥75%). Our average score of 2018 was Q30≥90%.

• Gene-expression analysis (raw data)
• Count table
• PCA plot, heatmap, pathway analysis