The breakthrough of extracellular RNAs (exRNAs) in biofluids has sparked a lot of curiosity about their biological functions and clinical applications. ExRNA biomarkers have been revealed as diagnostic, prognostic, and theranostic for a variety of diseases. Few have been verified across studies, most likely due to methodological differences between studies, such as exRNA isolation and measurement techniques. To improve the reproducibility of exRNA biomarker studies, a thorough understanding of the variability associated with these procedures is required.
Biofluids have also been found to contain small RNA biotypes other than miRNAs. Piwi-interacting RNAs (piRNAs) are 23- to 30-nt RNAs associated with transposon and other targets transcriptional and post-transcriptional silencing in germ cells. Human seminal fluid plasma, blood plasma, saliva, and urine have all been found to contain sequences that map to piRNAs. As a result, biofluid small RNA-seq allows for the unbiased discovery of disease-specific exRNA biomarkers. Recent studies have shown that frequently used small RNA-seq methods on exRNA and other low-input samples have high intra- and inter-lab reproducibility, as well as the wide acceptance of small RNA-seq for exRNA biomarker studies.
The three primary stages in the small RNA-seq workflow are I RNA isolation, (ii) cDNA library construction, and (iii) sequencing. Small RNA can be extracted using traditional total RNA extraction methods, then separated by length or bound to specific proteins. Because small RNA enrichment can result in losses and/or isolation-specific biases, total RNA analysis is a viable alternative and usually preferred technique. Reverse transcription (RT) of small RNAs produces complementary DNA (cDNA) for amplification and sequencing, which is then used to build the library. Because miRNA molecules are short, they are extended through ligation or polyadenylation, which introduces primer-binding sites for reverse transcription and subsequent amplification. In small RNA-seq, the extension step, particularly the ligation, is thought to be the most significant source of bias. Adaptor affinity for target molecules varies, causing artificial changes in true small RNA abundances, which throws the analysis off. Another source of bias is the amplification of cDNA molecules, which is caused by the PCR's various efficiencies when amplifying molecules of different lengths and secondary structures. This causes artificial shifts in the detected signals once more. In the field of mRNA sequencing, this PCR bias is well-known, and it is usually mitigated by using distinct molecular identifiers.
The discovery of extracellular RNAs (exRNAs) in biofluids has inspired a wave of curiosity about their biological functions and clinical applications. ExRNA biomarkers have been revealed as diagnostic, prognostic, and theranostic for a range of diseases.
The first implementation was in 2005 when Arabidopsis thaliana small RNA-seq was used to characterize various types of small RNAs. Small RNA-seq detected 18 new miRNAs in Caenorhabditis elegans a year later. Since then, over 700 human miRNA expression profiling studies have been published using small RNA-seq and have been deposited on Gene Expression Omnibus. It is now possible to evaluate miRNAs in individual cells thanks to the development of single-cell RNA-seq.
Some have been substantiated across studies, most likely due to methodological differences between studies, such as exRNA isolation and measurement techniques. As a result, a thorough understanding of the variability associated with these procedures is required for enhancing the reproducibility of exRNA biomarker experiments.