Found in all human biofluids, extracellular nucleic acids contain a lot of information about health and disease status. Liquid biopsies have been considered a leading powerful tool in the fight against cancer, in addition to forming non-invasive prenatal testing of fetal nucleic acids in maternal plasma. While studies using total RNA sequencing on biofluids have been effective, studies using small RNA sequencing and targeted or capture sequencing of longer RNAs have been uncommon. There have only been a few attempts at whole transcriptome profiling on urine, plasma, or extracellular vehicles (EVs), evaluating both polyadenylated and non-polyadenylated RNA transcripts. However, each of these techniques has flaws, such as short fragment length, low number of quantified genes, or high ribosomal RNA contamination. A comprehensive review of data quality in the form of technical repeatability and quantitative accuracy is missing from most of them.
Advantages of Biofluid RNA Sequencing
Total RNA sequencing has numerous benefits. Indeed, detection is not restricted to a set of pre-defined targets or polyadenylated RNAs at their 3′ ends. Other RNA biotypes, such as circular RNAs, histone RNAs, and a significant fraction of long non-coding RNAs, can be distinguished in addition to polyadenylated mRNAs. Furthermore, comparing exonic and intronic reads allows for the study of post-transcriptional regulation.
The EVs of nucleic acids found in various biofluids are promising biomarkers for disease diagnosis, prognosis, therapy response, and disease monitoring. Total RNA sequencing of biofluid specimens enables the investigation of posttranscriptional regulation and provides a more detailed understanding of the transcriptome. It can detect a variety of RNA biotypes, such as circular RNAs, histone RNAs, and long noncoding RNAs, as well as predefined targets and polyadenylated RNAs.
Challenges in Biofluid RNA Sequencing
ExRNA is a fascinating new branch of functional biomarker research, but still in its early stages and faces numerous technical challenges. RNA concentrations in biofluids are substantially lower than in tissue, and standard RNA extraction and quantification methodologies established for tissue are not always relevant. Another challenge is that the RNAs may be visible in various compartments, each with its own susceptibilities and vulnerabilities to RNA isolation techniques, resulting in an unintended bias in RNA profiling due to the isolation method used. Furthermore, samples made available to researchers are frequently not collected for exRNA analysis and are instead archived for certain lengths of time. Finally, little is known about exRNAs’ normal physiology or the degree of variation among healthy donors. All these variables must be taken into account when developing standard protocols for the study of exRNA.
Methods and Technologies for Biofluid RNA Sequencing
qRT-PCR, RNA microarray, and RNA sequencing are some of the current technologies for quantifying exRNA. While qRT-PCR and RNA microarrays use specific primers or probes to detect known RNA sequences, high throughput RNA-Seq can detect novel transcripts over a wide dynamic range, making it a potentially sensitive method for characterizing and quantifying exRNA. However, concerning the technique’s biases, the landscape of exRNAs, in biofluids like plasma, appears to be dominated by certain RNA species, such as ribosomal RNA fragments or specific miRNAs, resulting in a skewed distribution of exRNA species, thus limiting the sensitivity of detection of less abundant species.
CD Genomics offers transcriptomics services to meet the research objectives and budget of our customers, addressing the need to study mRNA, rRNA, tRNA, and other noncoding RNA isolated from cells or biofluids. CD Genomics has harnessed the power of next-generation sequencing and long-read sequencing to create an affordable, reliable, and high-throughput strategy from project design, project initiation, to high-quality results.