LncRNAs are conventionally classified as transcripts longer than 200 nt with no or low coding potential [24,25,26]. Similar to protein-coding transcripts, the transcription of lncRNAs is dependent on histone-modification-mediated regulation, and lncRNA’s transcripts are processed by the canonical spliceosome machinery. Overall, lncRNA genes show fewer exons than mRNAs, and appear to be under a weaker selective pressure during evolution. Moreover, some lncRNAs are expressed at levels lower than those of mRNAs and in a more tissue- and cell-specific manner, while others are known to be fairly abundant and are expressed in diverse cell types, such as the “house-keeping” genes [27].

Of tens of thousands of metazoan lncRNAs discovered from cDNA libraries and RNAseq data by high throughput transcriptome projects, only a handful of lncRNAs have been functionally characterized. The investigations on this small cohort of lncRNAs have demonstrated that these noncoding transcripts can serve as scaffolds or guides to regulate protein–protein or protein–DNA interactions [28,29,30,31] or can modulate post-translational modification of nonhistone proteins [32]. Moreover, lncRNAs are capable of controlling microRNAs (miRNAs) [33,34,35], and function as enhancers to influence gene transcription, when transcribed from the enhancer regions (enhancer RNA) [36,37,38] or their neighboring loci (noncoding RNA activator) [39,40].

Several lines of evidence have shown that lncRNAs are capable of influencing different cellular functions that are critical to tumorigenesis, such as cell proliferation, differentiation, migration, immune response, and apoptosis [41,42,43,44,45,46,47]. Furthermore, lncRNAs have been found to act as tumor suppressors or oncogenes [48,49,50,51], and, of note, a number of lncRNAs have been reported to be significantly deregulated in tumors [52,53,54,55].

The precise mechanism of lncRNAs release into the extracellular environment is not completely understood. Hypotheses have arisen that tumor cells, cancer-adjacent normal cells, immune cells, and other blood cells may all release lncRNAs [56,57], as shown in Figure 1. A few studies reported that lncRNAs can be encapsulated into membrane vesicles, such as exosomes or microvesicles (EV), prior to being secreted extracellularly. In such a conformation, the circulating lncRNAs have shown a higher degree stability, probably due to EVs offering protection against the nuclease-mediated degradation that may occur in the extracellular space and in body fluids [20,58,59] (Figure 1). On the other hand, other studies have suggested that the secretion pathway of lncRNAs may also occur in a similar manner to that for miRNAs. As such, lncRNAs might also be released into body fluids in an EV-independent fashion as complexes with high-density lipoproteins (HDLs) or protein Argonaute 2 (AGO2) [60] (Figure 1).

The hypothesis of an EV-independent mechanism for lncRNAs secretion might seem less likely given the high abundance of ribonucleases in serum, plasma, and other bodily fluids that can dramatically affect the stability of lncRNAs in the extracellular environment. However, one can speculate that circulating lncRNAs can be capable of resisting the RNase activity through modifications such as methylation, adenylation, and uridylation [61] or via the formation of higher order structures [62].

Difference sources of liquid biopsy (i.e., whole blood, plasma, serum, urine, and gastric juice) can be used to quantify circulating lncRNAs. However, due to the possibility of blood cell RNA contamination, whole blood is the less recommended option so far [63]. In addition, EDTA-anticoagulant collecting tubes have been suggested to be more suitable for the analysis of circulating lncRNAs [57]. Of note, some studies have found that lncRNAs remained stable in plasma even under multiple cycles of freeze–thaw, incubation at 45 °C, or storage at room temperature for as long as 24 h [56].

Overall, the methods to extract circulating lncRNAs can be divided into two major groups: guanidine/phenol/chloroform-based and column-based protocols. The column-based method is currently considered more reliable, since organic and phenolic contaminants in TRIzol-based methods might invalidate results [64].

Regarding the measuring and normalization methods, some studies have suggested that the use of an equal volume of input RNA sample may be more accurate than an equal amounts of RNA measured using a NanoDrop spectrophotometer since many diseases, including cancer, may indeed release an higher degree of RNAs into body fluids than healthy control groups, leading to a significantly higher level of circulating RNA in cancer patients that causes misleading results [64].

To date, quantitative real-time PCR (qRT-PCR) is still considered the gold standard for quantitative expression analysis of lncRNAs, including circulating lncRNAs [65]. Microarrays and whole transcriptome analysis (RNA-seq) still have limited uses in this field. In fact, the high throughput potential of microarrays relies on a reference database of targets, which in the case of circulating lncRNAs, is still very limited [21]; the RNA-seq requires huge amounts of starting RNA samples. Additionally, RNA-seq is currently expensive and needs special equipment and/or expert bioinformaticians [64], whereas, the targeted-approach of qRT-PCR is still more accessible, and saves money and time. Accordingly, qRT-PCR can be divided into relative and absolute analyses. In relative quantification methods, the choice of endogenous controls is critical to properly normalize the expression levels. In this regard, it must be noted that no systematic evaluation of reference genes for serum lncRNA has yet been reported, posing some limitations for the relative qRT-PCR method in the analysis of lncRNAs from a liquid biopsy.