mRNA-seq with Poly(A) selection is the best choice when the aim is to analyse the expression of protein-coding genes in eukaryotic organisms. Total RNA-seq with rRNA depletion should be chosen if your project also involves lncRNA, transcripts without a poly(A) tail, circRNA, degraded material or the transcriptome of prokaryotic organisms.
Comparison of methods
| Criterion | mRNA-seq - Poly(A) | Total RNA-seq - rRNA depletion |
|---|---|---|
| Enrichment method | Selection of RNA with a poly(A) tail | Removal of ribosomal RNA |
| Main fraction analysed | Mature eukaryotic mRNA | mRNA and a broader range of non-coding RNAs |
| Protein-coding genes | Yes | Yes |
| lncRNA | Only transcripts with poly(A) | Broader range |
| Transcripts without poly(A) | Usually not | Yes |
| circRNA | No | Analysis possible with the appropriate option |
| Mature miRNA | No | Usually require separate small RNA-seq |
| Degraded material | Less suitable | Usually a better choice |
| Prokaryotic organisms | Not routinely used | Yes, with an appropriately selected rRNA depletion method |
| Amount of data required | Smaller | Larger |
| Cost | Usually lower | Usually higher |
When should you choose mRNA-seq with Poly(A) selection?
Choose Poly(A) if:
- your main aim is to analyse the expression of protein-coding genes,
- you plan to perform differential-expression analysis of genes (detection of DEGs),
- you want to analyse alternative splicing and mRNA isoforms,
- the material under study comes from a eukaryotic organism,
- your samples contain RNA with good integrity,
- you want a high proportion of reads assigned to mRNA.
Poly(A) selection uses oligo(dT)-coated magnetic beads that bind to the poly(A) tails present at the ends of most mature eukaryotic mRNAs. This enriches the library for protein-coding transcripts.
When should you choose Total RNA-seq with rRNA depletion?
Choose rRNA depletion if:
- you need a broad view of the transcriptome,
- you are analysing both mRNA and lncRNA,
- you are interested in transcripts without a poly(A) tail,
- you plan to analyse circRNA,
- the material is partially degraded, for example from archival or FFPE samples,
- you are analysing the transcriptome of bacteria or archaea,
- the sample contains RNA from both the host and microorganisms.
In this approach, rRNA, which can account for up to approximately 80-90% of the sample, is removed from total RNA. The remaining RNA fractions are retained and can be included during library preparation.
Sample Requirements
mRNA-seq - Poly(A)
Recommended parameters:
- material: total RNA,
- amount: above 0.5 µg,
- concentration: above 50 ng/µl,
- volume: above 20 µl,
- OD260/280: 1.8-2.2,
- RIN: above 7, or above 6 for plants.
Minimum parameters:
- amount: above 0.2 µg,
- concentration measured using Qubit: above 20 ng/µl,
- volume: above 20 µl,
- OD260/280: 1.7-2.5,
- RIN: above 4.5, or above 4 for plants.
Total RNA-seq - rRNA depletion
Recommended parameters:
- material: total RNA,
- amount: above 2 µg,
- concentration: above 100 ng/µl,
- volume: above 10 µl,
- OD260/280: 1.8-2.2,
- RIN: above 7, or above 6 for plants.
Minimum parameters:
- amount: above 500 ng,
- concentration measured using Qubit: above 80 ng/µl,
- volume: above 10 µl,
- OD260/280: 1.7-2.5,
- RIN: above 6.5, or above 6 for plants.
Requirements may vary depending on the organism, the quality of the material and the library preparation kit selected. Samples that do not meet the standard criteria, including low-input material, require individual assessment.
Approximate data output
The following options are available for standard mRNA-seq with Poly(A) selection:
- 6 Gb per sample,
- 10 Gb per sample,
- 15 Gb per sample.
For Total RNA-seq with rRNA depletion, the recommended amount of data is larger:
- 12 Gb per sample,
- 16 Gb per sample - the recommended option if the project also includes circRNA analysis.
The final amount of data depends on factors including the size and complexity of the transcriptome, material type, analysis objective and the expected sensitivity for detecting lowly expressed transcripts.
Common mistakes when choosing a method
Choosing Poly(A) for degraded material
Degradation may lead to the loss of the 3′ fragment and the poly(A) tail. Such transcripts will not be efficiently captured, which may lead to underestimation of their abundance. From fragmented molecules, mainly the 3′ ends attached to the poly(A) tail are captured, which increases 3′ bias.
Assuming that Total RNA-seq automatically includes miRNA
Standard Total RNA-seq does not replace dedicated small RNA-seq. To analyse mature miRNA and other small RNAs, you need a separate library preparation protocol - Small RNA-seq.
Choosing Poly(A) to analyse prokaryotes
Bacterial and archaeal mRNAs lack the stable poly(A) tails typical of mature eukaryotic mRNA. Such projects use an appropriate rRNA depletion method.
Underestimating the amount of data required
Total RNA-seq covers a more diverse pool of transcripts, so it usually requires greater sequencing depth than mRNA-seq with Poly(A) selection.
Using an unsuitable rRNA depletion kit
Depletion efficiency depends on the organism being studied. Before starting the project, confirm that the kit is suitable for the species in question, particularly for non-model organisms and multispecies samples.
Combining different methods in a single comparison
You should not directly compare samples prepared using Poly(A) selection with those prepared using rRNA depletion in a single gene expression analysis without accounting for the strong effect of the library preparation method.
Questions for the project brief
Before preparing a quotation and planning the experiment, gather the following information:
Plan your project
Describe your project - we will recommend a method and amount of data.
Selected projects and client publications
See which institutions we perform next-generation sequencing (NGS) for and explore scientific publications that draw on the data we have supplied.