Alida Biosciences Frequently Asked Questions

RNA modifications are crucial for regulating numerous aspects of RNA biology, including RNA-protein interactions, splicing, three-dimensional RNA structure, transcript stability, intracellular trafficking, and translation. They offer broad insights into dynamic regulatory mechanisms in cancer, neurological and cardiometabolic disease, cell differentiation, viral infection and other phenotypes relevant to human, animal and plant health.

Unlike DNA methylation, which is heritable, RNA modifications are more dynamic and often reversible. They can change in response to environmental signals, developmental cues, or stress. This flexibility allows cells to fine-tune gene expression rapidly and adapt to changing conditions.

EpiPlex platform enables the localization and quantification of both m6A and inosine in mRNA and other long noncoding RNA species in a simple, 1-day workflow. Unlike other methods, EpiPlex assay can be run with low RNA sample input accommodating even the most precious clinically relevant samples. Designed for use with short-read sequencing, EpiPlex data is analyzed by our accompanying EpiScout software in a simple, push-button analysis of NGS output files.

EpiPlex assay utilizes non-antibody binders for m6A and inosine that have been engineered to be highly specific for the modification of interest. Compared to antibody enrichment, EpiPlex assay demonstrates greater sensitivity and specificity enabling sites of interest to be measured with lower RNA input and sequencing depth requirements. Additionally, EpiPlex assay is available as a complete reagent and software package providing all the necessary reagents, guidance and compute resources to go from RNA to actionable data output.

Traditional chemical methods, like MS (mass spectrometry), are highly sensitive to a broad range of modifications but lack sequence context and require significant material. Other chemical methods are designed to specifically modify bases which are read out as signatures (e.g., SNPs or indels) providing sequence context (e.g., bisulfite sequencing for m5C). However, chemical treatment is often destructive to RNA, exhibits significant off-target signal and typically has high material and sequencing depth requirements.

Mass spectrometry and the EpiPlex assay are highly orthogonal approaches. Mass spectrometry enables global detection and quantification of multiple RNA modifications but requires complete digestion of RNA into single nucleotides. As a result, it loses all information about which transcripts or pathways are being modified. ELISA-based methods face a similar limitation, quantifying global levels of a specific modification (provided a reliable antibody exists), but they also lack gene-specific resolution.

In contrast, the EpiPlex platform enables transcriptome-wide mapping of individual RNA modifications at the gene level in a one-day assay. It uses non-antibody, engineered binders to enrich and sequence modified RNA fragments directly from biological samples, revealing which transcripts are modified and enabling pathway-level insights and differential analysis across conditions. While it does not offer absolute quantification, each sample includes a spike-in control that serves as an internal reference for relative quantification. The kit also includes all necessary bioinformatic tools for alignment, peak calling, and sample comparison. Learn more.

Enrichment-based approaches, like EpiPlex assay, remove unmodified sequences improving sensitivity, particularly for low stoichiometry sites, and reduces sequencing depth requirements. A key drawback of enrichment is the loss of absolute quantification of modification stoichiometry. However, through the use of spike-in standards, such as those included in the EpiPlex kit, relative quantification of modifications between samples can still be obtained.

Yes, each sample includes reagents for a solution control that is unenriched by modifications and provides high quality RNA-seq data. Learn more.

Technical reproducibility and sensitivity to gene expression, as measured by both universal human reference RNA and ERCC controls, is comparable to leading RNA-seq library kits sold by third parties. Learn more.

The spike-in controls serve two purposes: 1) Confirms the assay successfully enriches for modifications and 2) Normalizes the signal to account for technical and biological variation. The detection of the spike-ins are necessary for the EpiScout analysis pipeline to properly function and therefore must be added to your samples.

RNA must be purified and free from contaminating DNA. Purified RNA should be stored in low TE buffer or nuclease-free water.

We suggest sequencing the enriched and solution control libraries to 25 million paired-end reads each. Depending on the complexity of your libraries and genes/sites in question, you may be able to sequence more or less than this suggested target. Please refer to our technical note for more information.

EpiPlex assay supports both total and polyA selected RNA as inputs. If using total RNA, a ribosomal RNA (rRNA) depletion step will be necessary. For more information on the effect of RNA input quantity on total or poly(A)-enriched RNA, please refer to our RNA input technical note.

EpiPlex assay works with degraded samples such as RNA isolated from FFPE treated tissue. Depending on the length of the RNA, a modification to the workflow may be required. Please contact support@alidabio.com for more information on running FFPE RNA through the EpiPlex workflow.

The total RNA workflow will capture both mature messenger RNA (mRNA) and immature messenger RNA (pre-mRNA), mitochondrial RNA (mtRNA) and long non–coding RNA (lncRNA). Ribosomal RNA (rRNA) can also be captured but should be depleted as part of the workflow and not make it on the sequencer in substantial amounts.

If rRNA is depleted post-library completion, we require the use of the SEQuoia RiboDepletion Kit from Bio-rad. Ribosomal RNA depleted at the beginning of the workflow is compatible with a number of depletion kits and may be used in the EpiPlex assay with the Poly(A)-enriched RNA workflow.

No, EpiPlex UDI primer kits are specific to the EpiPlex Library configuration. UDI kits from other vendors are not compatible with EpiPlex libraries. EpiPlex index primer sequences and barcodes for the 24 UDI set and 96 UDI set are freely available if you prefer to have them synthesized by a third party vendor.

Yes, but as optimal loading concentrations often vary between library types, providing a designated lane for EpiPlex libraries is suggested to maximize throughput.

EpiPlex libraries have been validated to be compatible with NextSeq 1000/2000, NovaSeq 6000 and NovaSeq X from Illumina and Aviti from Element Biosciences. Other short-read sequencers that accept Illumina-style adapters may work but have not been validated by AlidaBio.