*3.3. Experimental Validation*

After discovering linear lncRNAs and circRNAs by databases or high throughput methods, experimental approaches can be used to study their expression and function (Table 1). RNA interference (RNAi), antisense oligonucleotides (AOs), and CRISPR systems have successfully been used to knock down lncRNAs [56–62,73]. However, RNAi and AOs may have non-specific and off-target effects. Further, Goyal et al. found only 38% of lncRNAs were safe to be targeted without deregulating neighboring genes by CRISPR applications [61]. It might be necessary to use multiple strategies to select the best gene silencing approach. Real-time quantitative reverse transcription-polymerase chain reaction (QPCR) is employed to validate the expression of linear lncRNAs and circRNAs [54]. The limitations of QPCR include the need for highly sensitive assays to detect low expression molecules and selecting appropriate endogenous control genes of similar low expression and transcript size. RNA in situ hybridization (ISH) is used to visualize and localize lncRNAs [54]. Holdsworth-Carson et al. performed RNA-seq, RT-PCR, and ISH in endometriosis samples. They found that the long intergenic non-protein coding RNA 339 (LINC00339) is localized in the nucleus of ectopic endometriotic lesions [74]. While ISH allows for the localization of lncRNA, it is generally not quantifiable. Newer technologies, including single-cell RNA-sequencing and spatial transcriptomics, are being used to quantify the expression and localization of protein-coding genes [31,75]. For example, spatial transcriptomics allows both the quantification of expression and localization, but the limited depth of sequencing at this time precludes the identification of low expression molecules [31]. Several groups have used approaches based on protein precipitation to detect key interactions of binding proteins with lncRNAs. Wang et al. used RNA immunoprecipitation to define the LINC00261/miR-132–3p/BCL2L11 regulatory networks [76]. In addition to functional studies, studies correlating expression, localization, and endometriosis phenotype (i.e., pain or infertility, anatomic location of disease, number of adhesions) may significantly impact the field.
