The scope of our research is to provide a detailed RNA FISH protocol that's providing a reference for scientists who want to perform RNA FISH experiments. Our research has established that RNA FISH can be used to localize IncRNA in human osteosarcoma cells. Our protocol provided a comprehensive experimental procedure and precautions of RNA FISH using IncRNA SNHG6 in human osteosarcoma cells as a reference.
To begin, after acquiring the long noncoding RNA of interest, incubate the hybridization buffer at 37 degrees Celsius for two hours. Next, dissolve the four optical density probe in 160 microliters of diethyl pyrocarbonate-treated deionized water away from the light. Prepare 200 microliters of the probe mixture for each well and set up a series of 50, 25, 12.5, and six micrograms per milliliter.
Denature the probe mix at 73 degrees Celsius for five minutes. Take a 12-well cell culture plate containing 143B cells on sterile glass cover slips. Remove the medium and wash twice for five minutes with PBS.
Place the cells on a shaker. Then remove the PBS and add 200 microliters of 100%ethanol to each well, fixing for 15 minutes at room temperature. Then remove the ethanol, add 200 microliters of 0.1%Triton X-100 to each well and incubate for 15 minutes at room temperature.
Remove 0.1%Triton X-100 and wash two times for five minutes with PBS. Next, remove PBS. Add 200 microliters of 2X sodium saline citrate or SSC buffer into each well and incubate for 30 minutes at 37 degrees Celsius.
Remove 2X SSC buffer. Add 200 microliters of 70%ethanol into each well and incubate for three minutes at room temperature. Discard the 70%ethanol, then add 200 microliters of 85%ethanol into each well and incubate for three minutes at room temperature.
Next, discard the 85%ethanol. Add 200 microliters of 100%ethanol to each well and incubate for three minutes and room temperature. Afterward, absorb and discard the 100%ethanol and allow the wells to dry.
Next, add 200 microliters of denatured probe mixture to each well. Then incubate at 37 degrees celsius overnight. The following day, remove samples from 37 degrees Celsius, discard the probe mixture, and add 200 microliters of preheated 0.4X SSC 0.3%Tween-20 buffer to each well and wash for two minutes at room temperature.
Remove the 0.4X SSC 0.3%Tween-20 buffer. Add 200 microliters of 2X SSC 0.1%Tween-20 buffer to each well and wash for two minutes at room temperature. Next, discard the 2X SSC 0.1%Tween-20 buffer, add 200 microliters of DAPI staining solution and stain for 20 minutes in the dark on a shaker.
After staining, discard the DAPI solution and wash it with PBS for two minutes at room temperature. Lastly, add 50 microliters of mounting medium containing gum to the slide and place the glass cover slip to fix the slide. Representative SNHG6 FISH images in human osteosarcoma cells are shown.
SNHG6 probe labeled with Cy3, which emits red fluorescence, showed that SNHG6 is mainly localized in the cytoplasm. A background color was observed when using a high concentration of probe.
