RNA Fluorescence In Situ Hybridization for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

Summary

The present protocol describes a method of RNA fluorescence in situ hybridization to localize the lncRNAs in human osteosarcoma cells.

Abstract

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition (EMT), migration, infiltration, and autophagy of cancer cells. Localization detection of lncRNAs in cells can provide insight into their functions. By designing the lncRNA-specific antisense chain sequence followed by labeling with fluorescent dyes, RNA fluorescence in situ hybridization (FISH) can be applied to detect the cellular localization of lncRNAs. Together with the development of microscopy, the RNA FISH techniques now even allow for visualization of the poorly expressed lncRNAs. This method can not only detect the localization of lncRNAs alone, but also detect the colocalization of other RNAs, DNA, or proteins by using double-color or multicolor immunofluorescence. Here, we have included the detailed experimental operation procedure and precautions of RNA FISH by using lncRNA small nucleolar RNA host gene 6 (SNHG6) in human osteosarcoma cells (143B) as an example, to provide a reference for researchers who want to perform RNA FISH experiments, especially lncRNA FISH.

Introduction

Our understanding of the human genome has been greatly expanded by recent advances in whole-genome technology. About 93% of the human genome can be transcribed into RNAs, but only 2% of the RNAs can be translated into proteins; the remaining 98% of RNAs that have no protein translation function are called non-coding RNA (ncRNA)¹. As a class of noncoding RNAs (ncRNAs), long ncRNAs (lncRNAs), containing over 200 nucleotides², have attracted increasing attention due to their involvement in many physiological and pathological processes of the cells, such as differentiation, cycle control, apoptosis, migration, and invasion^3,4,5. LncRNAs play their roles through various mechanisms, such as regulating chromatin structure and nuclear gene expression, controlling the mRNA splicing process, and posttranscriptional modification⁶. LncRNAs regulate the occurrence, development, and metastasis of malignancies at both the transcriptional and posttranscriptional levels. Transcriptional regulation is realized in the nucleus by affecting RNA transcription via binding to the chromosomal structures, while posttranscriptional regulation is realized in the cytoplasm by controlling the target genes via an endogenous competitive RNA (ceRNA) mechanism^{5, 7, 8}. CeRNA has revealed a new mechanism of RNA interaction, namely that lncRNAs can act as a sponge to adsorb miRNAs and inhibit the miRNA-mediated degradation of related target genes⁹. Therefore, the information regarding the subcellular localization of lncRNAs, whether a specific lncRNA is located in the cytoplasm or nucleus, is important to help identify their biological functions.

At present, lncRNA localization is mainly detected by two methods, one is by nucleus/cytoplasm fraction isolation assay, and the other is by RNA FISH. In the former, RNAs in the cytoplasmic and the nuclear fractions are extracted respectively, and then PCR amplification is performed with specific lncRNA primers to detect the ratio of lncRNAs in the cytoplasm and nucleus. The advantage of this method is time efficiency, while the disadvantage is that the actual lncRNA localization is not directly reflected by the relative proportion of lncRNAs in the cytoplasm and nucleus. RNA FISH can detect lncRNA localization in cells through the design of lncRNA-specific antisense chain sequences followed by labeling with fluorescent dyes¹⁰. The RNA FISH methods have been improved with advances in probe techniques and detection methods, including fluorophore-labeled multiple oligo probe sets¹¹, LNA probes¹², and branched-DNA (bDNA) probes¹³. RNA FISH can not only detect the localization of lncRNA, but also detect the colocalization of other RNAs, DNA, or proteins by using double-color or multicolor immunofluorescence¹⁴.

In this work, we have included the detailed intracellular localization detection protocol of lncRNA small nucleolar RNA host gene 6 (SNHG6) in osteosarcoma cells (143B) by RNA FISH as an example. SNHG6 is a 600-730 nucleotide lncRNA in its mature spliced form and identified as a novel oncogene in diverse human cancers, including colorectal cancer, gastric cancer, ovarian clear cell carcinoma, osteosarcoma, and hepatocellular carcinoma^15,16,17,18. Studies have confirmed the involvement of SNHG6 in biological behaviors of cancer cells, such as proliferation, EMT, and autophagy, and shown the cytoplasmic localization of SNHG6 where it may affect the target genes by binding (sponging) the miRNAs^15,16,17. This detailed detection protocol of SNHG6 intracellular localization by RNA FISH is presented herein.

Protocol

See the Table of Materials for details of all materials, reagents, and instruments used in this protocol. Figure 1 shows the overall protocol for RNA FISH; Table 1 contains the composition of all solutions and Table 2 contains the primer sequences used in this protocol.

1. Probe preparation

1. Identify and acquire the FASTA sequence of a target lncRNA of interest, for example, from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Following the indications on the website, design the ISH probes online¹⁹, and review the design algorithm's suggestions to list the probes to be ordered with Cy3 label.
2. Incubate the hybridization buffer at 37 °C for 2 h in advance.
3. Dissolve 4 optical density (OD) probe in 160 µL of diethylpyrocarbonate (DEPC)-treated ddH₂O at the concentration of 1 mg/mL away from light.
   NOTE: Optical density (OD) represents the measurement unit of DNA and RNA. Usually, 1 OD unit = 33 µg/mL DNA.
4. Prepare 200 µL of the probe mixture for each well (1.2 µL-10 µL of probe, 70 µL of hybridization buffer, make up the volume with DEPC-treated ddH₂O to 200 µL), and set up a series of 50 µg/mL, 25 µg/mL, 12.5 µg/mL, and 6 µg/mL.
   NOTE: The probe concentration needs to be explored experimentally in advance. A high probe concentration will lead to non-specific binding of lncRNAs, while a low probe concentration will lead to insensitive or failed detection of lncRNAs.
5. Denature the probe mix at 73 °C for 5 min.
   NOTE: If the Cy3-labeled DNA probe is double-stranded, denature the probe to single-stranded DNA at 95 °C for 5 min, then quickly cool for 2 min on ice.

2. Cell preparation

1. Seed 50,000 143B cells per well on sterile glass coverslips in a 12-well cell culture plate and incubate them for 24 h (37 °C, 5% CO₂) in DMEM.
   NOTE: The specific cell number seeded here varies with cell size, which is appropriate for the seeded cells to reach a confluence of 50% after being incubated for 24 h. The sterile glass coverslips are round to be suitable for the 12-well plate.
2. Remove the medium and wash for 2 x 5 min with 1x phosphate-buffered saline (PBS).
   NOTE: Prepare 1x PBS with DEPC-treated ddH₂O. PBS without DEPC-treatment cannot be used because it contains RNase. Perform all the following steps in RNase-free conditions.
3. Remove 1x PBS and add 200 µL of 100% ethanol in each well to fix for 15 min at room temperature.
4. Remove the ethanol, add 200 µL of 0.1% Triton X-100 (in 1x PBS) in each well, and incubate for 15 min at room temperature.
   NOTE: The time needs to be strictly controlled with Triton X-100 permeabilization and cannot be too long.
5. Remove 0.1% Triton X-100 and wash 2 x 5 min with 1x PBS.
   NOTE: If the protocol has to be paused here, replace the PBS with 70% ethanol (dilution of 100% ethanol with RNase-free ddH₂O) and store the sample at 4 °C for up to 3 months.
6. Remove 1x PBS, add 200 µL of 2x sodium saline citrate (SSC) buffer (dilution of 20x SSC with RNase-free ddH₂O) into each well, and incubate for 30 min at 37 °C.
7. Remove 2x SSC buffer, add 200 µL of 70% ethanol into each well, and incubate for 3 min at room temperature.
8. Discard the 70% ethanol, add 200 µL of 85% ethanol (dilution of 100% ethanol with RNase-free ddH₂O) into each well, and incubate for 3 min at room temperature.
9. Discard the 85% ethanol, add 200 µL of 100% ethanol into each well, and incubate for 3 min at room temperature.
10. Absorb and discard 100% ethanol; let the wells dry.

3. Fluorescence in situ hybridization (FISH)

1. Add 200 µL of probe mixture (denatured as in step 1.5) to each well and incubate at 37 °C overnight (16-18 h).
   NOTE: There is a strong positive correlation between hybridization temperature and probe concentration; therefore, to optimize the background, both the hybridization temperature and the probe concentration should be reduced.
2. The next day, take out samples from 37 °C and discard the probe mixture. Add 200 µL of 0.4x SSC/0.3% Tween-20 buffer to each well (preheated at 65 °C) and wash for 2 min at room temperature.
3. Remove the 0.4x SSC/0.3% Tween-20 buffer, add 200 µL of 2x SSC/0.1% Tween-20 buffer to each well, and wash for 2 min at room temperature.
4. Remove 2x SSC/0.1% Tween-20 buffer, add 200 µL of 4',6-diamidino-2-phenylindole (DAPI) staining solution (1 µg/mL), and stain for 20 min away from light.
5. Discard the DAPI dye solution and wash with 1x PBS for 2 min at room temperature.
6. Add 50 µL of mounting medium containing gum onto the slide and place the glass coverslip on the slide to fix.
   NOTE: Be sure to place the coverslip on the slide with the cell side down.
7. Observe under a fluorescence microscope.
   NOTE: Use a fluorescence microscope or a laser confocal microscope; the latter produces a higher sensitivity and clarity imaging.

Representative Results

Representative images of SNHG6 FISH in human osteosarcoma cells are shown (Figure 2). The negative control is treated with the negative Ctrl probe; positive control is treated with U6 probe ²⁰. SNHG6 probe and U6 probe are labeled with Cy3, which emits red fluorescence. DAPI is a dye that stains the DNA, which emits blue fluorescence. This result shows that SNHG6 is mainly localized in the cytoplasm, and this information can provide an important direction for further study of SNHG6.

If a very high concentration of probe is used, a background color can be seen under the fluorescence microscope. Figure 3 shows the representative images when a high concentration of SNHG6 probe is used in RNA FISH. The white arrows represent nonspecific staining.

Figure 1: Flow chart of the RNA FISH protocol for lncRNA. This chart shows the key protocol of the RNA FISH experiment process. Abbreviations: FISH = fluorescence in situ hybridization; lncRNA = long noncoding RNA. Please click here to view a larger version of this figure.

Figure 2: The localization of LncRNA (SNHG6) in human osteosarcoma cells (143B). Negative control is treated with a negative control probe. Positive control is treated with a U6 probe. SNHG6 and U6 probes are labeled with Cy3, which emits red fluorescence. DAPI is a dye that stains the DNA and emits blue fluorescence. Red and blue merge to make pink. The images are taken using a fluorescence microscope. Scale bar = 10 µm. Abbreviations: LncRNA = long non-coding RNA; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.

Figure 3: The representative images of high concentration of SNHG6 probe in RNA FISH. SNHG6 probes are labeled with Cy3, which emits red fluorescence. DAPI is a dye that stains the DNA and emits blue fluorescence. Red and blue merge to make pink. The images are taken using a fluorescence microscope. Scale bar = 10 µm. Please click here to view a larger version of this figure.

Table 1: The composition of solutions used in the RNA FISH hybridization experiment. All dilutions should be done with sterile, RNase-free water. All added water is DEPC-treated ddH₂O. Abbreviations: FISH = fluorescence in situ hybridization; DEPC = diethylpyrocarbonate. Please click here to download this Table.

Table 2: All probe sequences used in this experiment. Please click here to download this Table.

Discussion

This RNA FISH protocol can not only detect the localization of lncRNAs in cells, but also detect the colocalization of other RNAs, DNA, or proteins in cells, which can also be used to detect the location of lncRNAs in paraffin-embedded tissues. However, the specific protocol in such cases is different because the paraffin-embedded tissues need to be dewaxed²¹. This experimental procedure can be applied in 48- or 96-well plates, but 384-well plates are too small to be used here.

Several critical steps of this method need to be noted, including the RNase-free environment, probe concentration, hybridization temperature, and the setting of negative and positive controls. First, the RNase-free condition is crucial because the RNase breaks down the bonds between nucleotides and leads to the degradation of lncRNAs in general. Second, the commonly recommended probe concentration ranges from 5 to 50 µg/mL. A high probe concentration will lead to non-specific binding of lncRNAs, while a low probe concentration will lead to insensitive or failed detection of lncRNAs¹⁹. If a very high concentration of probe is used, a background color can be seen under the fluorescence microscope even in the presence of a scrambled probe (a negative control) only; therefore, the same concentrations of negative controls are usually used to set up the baseline (same as the internal control) during the preliminary experiments exploring the appropriate probe concentration to avoid any non-specific binding. The highest probe concentration without background color in the negative control group, presenting the strongest signaling without non-specific binding, was used in the following experiments. Third,it is recommended to design at least two or three probes for optimization with a specific lncRNA. At the same time, two or more probes can also be mixed to improve the detection sensitivity of target lncRNAs. Fourth, the normal hybridization temperature of the lncRNA probe is 37 °C. During the hybridization process, the hybridization temperature should be kept constant and uniform. The range of hybridization temperature between different probes is 34 to 38 °C. The hybridization temperature is strongly positively correlated with the probe concentration; therefore, to optimize the background, the probe concentration should be reduced. Lastly, it is also critical to set negative and positive controls. The group without probe is used as a negative control to obtain background signals. A U6 probe or a ribosomal RNA probe is used as a positive control to rule out the possibility of false positive results.

This approach has several advantages. The fluorescence color of lncRNA probes can be changed. Green fluorescence is emitted by fluorescein isothiocyanate (FITC), fluorescein (FAM), and Alexa Fluor 488, while red fluorescence is emitted by Cy3 and Alexa Fluor 555. FITC is used for typical green and Cy3 for typical red. In addition, the images can be obtained by using either a fluorescence microscope or a laser confocal microscope. The latter's images are more sensitive and clearer than those acquired by using a fluorescence microscope.

The limitation of the RNA FISH method is that this is a qualitative method; therefore, the results are not quantifiable. Due to the change in hybridization time and the influence of subjective factors during fluorescence microscope observation, lncRNA expression cannot be accurately quantified in cells. The expression levels of lncRNAs in different cellular fractions can only be qualitatively evaluated. However, with improvement in the technique, a single-molecule fluorescent in situ hybridization (smFISH) method²² has been developed to quantify the RNA expression level; more smFISH detection may be reported in the future.

The RNA FISH technique has a wide range of applications. The intracellular localization detection of lncRNAs will benefit the study of the biological mechanisms of lncRNAs. At the same time, this technique can also be used to detect the localization of other non-coding RNAs in cells, including circRNAs, miRNAs, and tRNAs.

Materials

Name	Company	Catalog Number	Comments
Automatic cell counter	Shanghai Simo Biological Technology Co., Ltd	IC1000	Counting cells
Cell culture plate-12	Shanghai YueNian Biotechnology Co., Ltd	3513,corning	Place the coverslips in the plate
Cell line (143B)	Cell Bank of Chinese Academy of Sciences	CRL-8303	osteosarcoma cancer cell line
Centrifuge tube (15 mL, 50 mL)	Shanghai YueNian Biotechnology Co., Ltd	430790, Corning	Centrifuge the cells
Coverslips	Shanghai YueNian Biotechnology Co., Ltd	abs7026	The cells are seeded on the coverslips
Cy3 label-SNHG6 DNA probe	Shanghai GenePharma Co.,Ltd	A10005	Detect SNHG6 location
DMEM media	Shanghai YueNian Biotechnology Co., Ltd	LM-E1141	Cell culture medium
Dry Bath Incubator	Haimen Kylin-Bell Lab Instruments Co.,Ltd.	DKT200-2	Incubation at different high temperatures
Ethanol 100%	Sinopharm Chemical ReagentCo., Ltd	10009218	dehydration
Fluorescence microscope	Shanghai Waihai Biotechnology Co., LTD	Olympus BX43 equipped with a camera of Olympus U-TV0.5XC-3(SN:5J01719),olympus	Observation and positioning
Incubator	Shanghai Yiheng Scientific Instrument Co., LTD	DHP-9051	The samples were incubated at 37 °C.
Mounting Medium	Sangon Biotech (Shanghai) Co., Ltd.	E675004	Attach the coverslips to the slide
Shaker	Haimen Kylin-Bell Lab Instruments Co.,Ltd.	TS-8S	Washing sample
Slide	Shanghai YueNian Biotechnology Co., Ltd	188105	The coverslips is placed on the slide
Triton X-100	Sangon Biotech (Shanghai) Co., Ltd.	A600198	Permeable membrane and nuclear membrane
Trypsin (0.25%)	Shanghai YueNian Biotechnology Co., Ltd	25200056, Gibco	trypsin treatment of cells
Tween-20	Sangon Biotech (Shanghai) Co., Ltd.	A600560	detergent