tiRNA and tRF Sequencing

Transfer RNA (tRNA) is a molecule that decodes mRNA and translates proteins. tRNA produces two degradation products through a precise biogenesis process: tiRNA (tRNA halves) and tRF (tRNA-derived fragments). In recent years, next-generation sequencing offers unprecedented opportunities to discover and quantify a wide variety of tRFs and tiRNAs. RNA-seq is an effective tool for high-throughput detection of tRF and tiRNA expression, and researchers has designed a variety of specific amplification primers to verify them in the database by qRT-PCR.

Fig 1. tRF & tiRNA-seq experiment workflow. (Fang Y, 2021)

Applications of tRF and tiRNA

In general, tRFs and tiRNAs exert their biological functions by regulating translation, gene expression, mRNA stability, epigenetics, and chromatin modification.

• Biological Functions: Acting as microRNAs in RNA interference, regulating the stability of target mRNAs.
• Human Disease Research: tRFs and tiRNAs are associated with a variety of disease conditions such as cancer, neurodegenerative and metabolic disorders.
• Biomarker Potentials: The ratio of tRFs/tiRNAs has been applied as a good indicator of cancer progression-free survival and a candidate prognostic marker.

Fig 2. tRFs and tiRNAs regulate translation. (Xie Y, 2020)

Our Services

BOC Sciences provides tRF&tiRNA sequencing services to offer insights into the study of biological functions and their underlying mechanisms. We are committed to helping our clients advance their next steps in RNA research. With the expertise in tRF&tiRNA, BOC Sciences tRFs&tiRNA sequencing service offers:

• Identification and analysis of tRF&tiRNA, and prediction of their target genes.
• Qualitative and quantitative analysis based on comprehensive tRF and tiRNA collections from our in-house database.
• Bioinformatics analysis:

1. Differential analysis data with detailed annotation of tRF&tiRNA sequences, types, lengths and cleavage sites in tRNAs.
2. Distribution of tRF and tiRNA subtypes with an accurate annotation and classification system based on tRNA topology and statistical significance.

tiRNA and tRF Sequencing Workflow

• Sample Preparation

1. Grouping of experimental mice
2. Radiation damage model construction
3. Tissue collection and preservation
4. Hematoxylin and eosin (H&E) staining

• RNA Extraction and Quality Control

Quality control is performed using gel electrophoresis (GC) to identify the quantity and integrity of each RNA sample, followed by a spectrophotometer for total RNA quantification and quality assurance.

• tRF & tiRNA Pretreatment

Pretreatment of total RNA samples to remove RNA modifications that may interfere with small RNA-seq library construction.

• Library Preparation

The sequencing library is constructed by using a commercial kit, which included 3' and 5' small RNA adapters, cDNA synthesis and library PCR amplification. Therefore, the influence of miRNA and other small RNA fragments can be excluded. At BOC Sciences, the prepared tRF and tiRNA-seq libraries are fully quantified.

• Sequencing

Sequencing reactions are performed on based on the manufacturer's guide.

• Data Collection and Analysis

We evaluate tRF and tiRNAs levels using sequencing counts. tRF and tiRNA differentially expressed are screened based on calculated values using the R package EdgeR.

• Target Gene Prediction

Depending on the miRNA target predictors, our experts use different algorithms to obtain the seed sequences and targets for tRFs and tiRNAs.

• Bioinformatics Prediction

BOC Sciences provides gene annotation and analysis services, which can be used for pathway and process enrichment analysis of target genes.

1. Protein Interaction Enrichment Analysis
2. Molecular Complex Detection (MCODE) Algorithm
3. Verify the enrichment of target genes

• RT-qPCR

We perform RT-qPCR verification for candidate tRFs and tiRNAs.

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tiRNA and tRF Sequencing

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