Total RNA sequencing captures both coding and non-coding RNAs by removing rRNA instead of enriching poly(A) RNA. This approach enables comprehensive transcriptome analysis, including mRNA, lncRNA, circRNA, and other regulatory transcripts.
This makes Total RNA-Seq increasingly important in systems biology, disease research, and transcriptome discovery.
Major Application Areas
Total RNA library preparation is widely used for:
|
Application |
Description |
|
Whole transcriptome profiling |
Analyze coding and non-coding RNA |
|
Non-coding RNA discovery |
Identify novel regulatory RNAs |
|
Host–pathogen studies |
Explore infection-related transcript changes |
|
Degraded sample sequencing |
Suitable for FFPE RNA samples |
Because it retains a broader RNA population, this approach is particularly valuable for exploratory transcriptomics.
Industry Pain Points
Researchers performing Total RNA-Seq often face challenges such as:
|
Challenge |
Impact |
|
High rRNA background |
Wastes sequencing reads |
|
Complex depletion protocols |
Increases workflow time |
|
Low-abundance transcripts |
Reduced detection sensitivity |
Since rRNA accounts for 80–90% of total RNA, efficient removal is essential.
Yeasen Total RNA Library Preparation Workflow
Yeasen provides comprehensive total RNA library preparation workflows, offering both strand-specific and non-strand-specific library construction options in a single system. In addition, the premixed total RNA library preparation kit simplifies experimental steps and streamlines the workflow, enabling fast and efficient library preparation.
Typical workflow includes:
The optimized protocol improves informative sequencing reads and transcript coverage.
Example Case Study
Whole-Transcriptome Sequencing Reveals Dynamic mRNA and ncRNA Regulation in Osteosarcoma Progression
Using deep whole-transcriptome sequencing, distinct expression patterns of miRNAs, lncRNAs, and mRNAs were successfully characterized during osteosarcoma (OS) tumorigenesis and pulmonary metastasis.
Results demonstrated:
- Clear separation of primary OS, metastatic OS, and normal controls by PCA analysis
- Identification of 65 DEmiRNAs, 233 DElncRNAs, and 1,405 DEmRNAs in primary OS versus controls
- Detection of 48 DEmiRNAs, 50 DElncRNAs, and 307 DEmRNAs in pulmonary metastatic OS versus primary OS
- Distinct hierarchical clustering patterns across miRNA, lncRNA, and mRNA expression profiles
- Robust differentiation between disease stages through unsupervised clustering analysis
Figure 1. Whole-transcriptome sequencing revealed distinct miRNA, lncRNA, and mRNA expression patterns during osteosarcoma progression and pulmonary metastasis, with clear separation between tumor and control groups by PCA and clustering analysis.
Adapted from: Xie L, et al. Cell Death & Disease, 2018.
These findings revealed the dynamic regulation of coding and non-coding RNAs during osteosarcoma progression and metastasis, supporting the value of integrated transcriptome sequencing for biomarker discovery and cancer mechanism research.
Total RNA / Whole-Transcriptome Library Preparation Tips
|
Factor |
Recommendation |
|
RNA quality |
High-quality RNA improves detection |
|
Strand specificity |
Important for lncRNA annotation |
|
Sequencing depth |
50–100 M reads recommended |
|
Bioinformatics Analysis |
Use integrated pipelines for simultaneous mRNA and ncRNA profiling |
FAQ
Q1. What is total RNA sequencing?
Total RNA-seq captures all RNA species in a sample after rRNA removal, including mRNA, lncRNA, circRNA, and other non-coding RNAs.
Q2. When should I choose total RNA-seq instead of mRNA-seq?
Total RNA-seq is recommended when studying global transcriptome profiles, RNA processing, or when investigating coding and non-coding RNA simultaneously.
Q3. Does total RNA-seq require more sequencing depth?
Yes. Because total RNA-seq includes a broader range of RNA species, it often requires higher sequencing depth compared to mRNA-seq.
Q4. What samples are suitable for total RNA-seq?
Total RNA-seq works well for degraded samples, FFPE tissues, and complex transcriptome studies, especially when rRNA depletion strategies are used.
Related Product
|
Category |
Name |
Cat. No. |
Size |
|
|
RNA Lib Prep |
Dual-mode(Strand specific & Non Strand specific) |
12308ES24/96 |
24 T/96 T |
|
|
Premix version |
12340ES24/96 |
|||
|
12341ES24/96 |
||||
|
mRNA isolation |
Eukaryotic mRNA |
12629ES24/96 |
24 T/96 T |
|
|
rRNA depletion |
Human/Mouse/Rat |
Hieff NGS™ MaxUp Human/Mouse/Rat rRNA Depletion Kit(rRNA ITS/ETS) |
12257ES24/96 |
|
|
Hieff NGS™ MaxUp Human/Mouse/Rat rRNA Depletion Kit(rRNA ITS/ETS) 2.0 |
12726ES24/96 |
|||
|
Plant |
12254ES24/96 |
|||
|
Beads |
- |
12602ES03/08/56 |
1/5/60 mL |
|