The miND® spike-in has been developed for small RNA sequencing experiments and absolute quantitation of microRNAs in any biological matrix and species, overcoming previous challenges of quality control and difficulty in achieving absolute quantitation of miRNAs across different biological matrix and species (Khamina et al, 2022).
miRNAs are small endogenous non-coding RNAs of 17–25 nucleotides in length that regulate gene expression in mammalian cells. miRNAs are produced by virtually all cell types, and their expression can be changed in response to physiological stimuli and pathological processes. Multiple studies indicated that levels of microRNAs can be used as diagnostic and prognostic biomarkers for various diseases.
Small RNA sequencing is a commonly used NGS technology for identification of various types of short non-coding RNAs, including microRNAs in tissues and biofluids. The detection of miRNA by NGS can be affected depending on the technical methods used for library preparation, which will bias the relative abundance of the selected microRNAs across different samples.
TAmiRNA has now developed the miND® spike-in as a commercial product (Cat no: KT-041-MIND) that consists of seven unique core sequences mixed in specific proportions to cover a wide range of endogenous microRNAs, representing up to 65,536 possible 21 nucleotide (nt) sequences. Each unique core sequence consists of 13 nts that do not match the genome of interest, flanked by a set of four randomized nts on both 5’ and 3’ ends to reduce sequencing bias and ensure precise quantitation of small RNA. Altogether, the miND® spike- contains up to 458,752 unique oligonucleotides.
The product is designed to be simply added to an RNA sample during the library preparation.
The miND® spike-in sequences are detected in the NGS data along with the endogenous small RNAs. Read counts of the miND® spike-in and endogenous miRNAs are used to calculate absolute concentrations (amol/μL or molecules/μL). This conversion can either be achieved by using TAmiRNA’s miND® NGS data pipeline (Diendorfer et al. 2022) or by incorporating provided scripts in an already established NGS data analysis workflow.
The miND® technology developed as part of TAmiRNA’s participation in the international IMI2 project Translational Safety Biomarker Pipeline (TransBioLine) focused on the development robust assay liquid biopsies using non-invasive miRNA biomarkers.
TAmiRNA has filed patent for miND® under WO2018138334A1 related to novel spike-in oligonucleotides for absolute quantitation of nucleotide sequence data and quality control across different sample types and patients populations.
miND® spike benefits
The miND® spike-in simultaneously enables quality control and absolute quantitation of microRNAs across different sample types, delivering a series of specific benefits:
- Serviceable quality control for small RNA-sequencing experiments to confirm the dynamic range and sensitivity of the assay
- Ability to generate a linear regression model to calculate absolute concentrations of endogenous miRNAs across different sample types, biological conditions and datasets
- Presence of companion TAmiRNA NGS data analysis pipeline that processes NGS raw data and presents the QC overview of the NGS data in a comprehensive html report. The pipeline performs the conversion of read counts for endogenous small RNAs to the absolute concentrations (amol or molecules/μL)
- Fit-for-purpose validation in the context of RealSeq®-Biofluids Plasma/Serum miRNA Library Kit for Illumina® sequencing
- Khamina, K., Diendorfer, A.B., Skalicky, S., Weigl, M., Pultar, M., Krammer, T.L., Fournier, C.A., Schofield, A.L., Otto, C., Smith, A.T., Buchtele, N., Schoergenhofer, C., Jilma, B., Frank, B.J.H., Hofstaetter, J.G., Grillari, R., Grillari, J., Ruprecht, K., Goldring, C.E. and Rehrauer, H. (2022). A MicroRNA Next-Generation-Sequencing Discovery Assay (miND) for Genome-Scale Analysis and Absolute Quantitation of Circulating MicroRNA Biomarkers.International Journal of Molecular Sciences, [online] 23(3), p.1226. doi:10.3390/ijms23031226.
- Diendorfer, A., Khamina, K., Pultar, M. and Hackl, M. (2022). miND (miRNA NGS Discovery pipeline): a small RNA-seq analysis pipeline and report generator for microRNA biomarker discovery studies. [online] f1000research.com. Available at: f1000research.com/articles/11-233/v1.
- Gutmann, C., Khamina, K., Theofilatos, K., Diendorfer, A.B., Burnap, S.A., Nabeebaccus, A., Fish, M., McPhail, M.J.W., O’Gallagher, K., Schmidt, L.E., Cassel, C., Auzinger, G., Napoli, S., Mujib, S.F., Trovato, F., Sanderson, B., Merrick, B., Roy, R., Edgeworth, J.D. and Shah, A.M. (2021). Association of cardiometabolic microRNAs with COVID-19 severity and mortality. Cardiovascular Research, [online] 118(2), pp.461–474. doi:10.1093/cvr/cvab338.
- Lutzmayer, S., Enugutti, B. and Nodine, M.D. (2017). Novel small RNA spike-in oligonucleotides enable absolute normalization of small RNA-Seq data. Scientific Reports, 7(1). doi:10.1038/s41598-017-06174-3.
Click on TAmiRNA miND spike-ins for further information on the product.
Click on A MicroRNA Next-Generation-Sequencing Discovery Assay (miND) for Genome-Scale Analysis and Absolute Quantitation of Circulating MicroRNA Biomarkers to access original research paper lead-authored by Dr. Kseniya Khamina