Evaluation of fluorescence in situ hybridization techniques to study long non-coding RNA expression in cultured cells

Abstract

Deciphering the functions of long non-coding RNAs (lncRNAs) is facilitated by visualization of their subcellular localization using in situ hybridization (ISH) techniques. We evaluated four different ISH methods for detection of MALAT1 and CYTOR in cultured cells: a multiple probe detection approach with or without enzymatic signal amplification, a branched-DNA (bDNA) probe and an LNA-modified probe with enzymatic signal amplification. All four methods adequately stained MALAT1 in the nucleus in all of three cell lines investigated, HeLa, NHDF and T47D, and three of the methods detected the less expressed CYTOR. The sensitivity of the four ISH methods was evaluated by image analysis. In all three cell lines, the two methods involving enzymatic amplification gave the most intense MALAT1 signal, but the signal-to-background ratios were not different. CYTOR was best detected using the bDNA method. All four ISH methods showed significantly reduced MALAT1 signal in knock-out cells, and siRNA-induced knock-down of CYTOR resulted in significantly reduced CYTOR ISH signal, indicating good specificity of the probe designs and detection systems. Our data suggest that the ISH methods allow detection of both abundant and less abundantly expressed lncRNAs, although the latter required the use of the most specific and sensitive probe detection system.

Super-Enhancer–Associated Long Noncoding RNA HCCL5 Is Activated by ZEB1 and Promotes the Malignancy of Hepatocellular Carcinoma

“…We next performed the nuclear/cytosol fractionation experiment to determine the subcellular localization of HCCL5. With validating the nuclear/cytosol fractionation successfully, lncRNA MALAT1 was located in the nucleus (35)(36)(37)(38), while both MT-CO1 mRNA (Mitochondrially Encoded Cytochrome C Oxidase I) and GAPDH mRNA were mainly located in the cytoplasmic faction. Importantly, the majority of HCCL5 was distributed in cytoplasm ( Supplementary Fig.…”

Long noncoding RNA LINC00261 suppresses prostate cancer tumorigenesis through upregulation of GATA6-mediated DKK3

“…The subcellular localization of LINC00261 in prostate cancer cells was identified by FISH, and the results were analyzed by the Biological Prediction Website. According to the instructions of Ribo ™ lncRNA FISH Probe Mix (Red) (Guangzhou RiboBio Co., Ltd., Guangdong, China), the specific methods were as follows [14]: cells were cultured into a 24-well plate at 6 × 10 4 cells/well until they reached 80% confluence. Next, the cells were fixed with 1 mL 4% polyformaldehyde.…”

Single molecule chromogenic in situ hybridization assay for RNA visualization in fixed cells and tissues

“…We performed detection of long noncoding RNA (lncRNA) MALAT1 and CYTOR in cultured cancer cells H1299 and SW480 to test if we can detect the subcellular location of RNA molecules. MALAT1 and CYTOR are previously known to be mainly expressed in the nucleus and cytoplasm (Soares et al 2017). As shown in Figure 5, we can see that most of the MALAT1 are detected in the nuclei and there are more CYTOR located in the cytoplasm than that in the nuclei.…”