The overall goal of the following experiment is to demonstrate sensitive and specific detection of high risk HPVE six, E seven mRNA in tumor cells in head and neck cancers by using a novel RNA in situ hybridization technique called RNA scope. This is achieved by first pretreating, the tissue samples on the slides with heat and protease to perme the cells to allow probe access. As a second step incubate slides with an HPV probe cocktail targeting seven high risk HPV types, which hybridizes to E six E seven mRNA.
Next, amplify hybridization signals by hybridizing sequentially with the pre amplifier amplifier and HRP conjugated label probe. The signal is then detected with a chromogen and visualized under standard brightfield microscopy results are obtained that show HPVE six E seven mRNA signals as Punctate brown precipitants in HPV positive tumors, but not in HPV negative tumors. The RRA scope assay uses a novel and proprietary probe design strategy for insect hybridization.
It allows single molecule visualization and target quantification. This technology paves the way for researchers in the field of biomarker research and the diagnostic acid development to identify and validate any RNA biomarkers discovered through macro race or next generation sequencing studies. Most scientists with previous experience in IHC or each can pick up this method quickly.
Now I will give you a demonstration of the entire workflow highlighting the key steps to help you master the technique. The tissue specimens to be assayed by this RNA in situ hybridization technique are formal and fixed. Paraffin embedded sections of five plus or minus one micron thickness mounted on slides one hour prior to the assay.
Bake the slides in a dry oven at 60 degrees Celsius while the slides are baking. Prepare the hybridization oven. Bring the temperature to 40 degrees Celsius and place a humidifying paper in the humidity control tray.
Add 50 milliliters of distilled water to the humidifying paper to wet it completely. Insert the covered tray into the oven to prewarm for at least 30 minutes before use. Prepare 700 milliliters of one x pretreat two solution by diluting 10 x pretreatment solution in distilled water.
Heat the one x pretreat two solution to boiling and maintain the temperature between 100 and 104 degrees Celsius. While preventing over boiling. Make three liters of one x wash buffer by diluting prewarm 50 x wash buffer in distilled water under a fume hood.
Prepare the xylenes, 70%ethanol and 100%ethanol for de depa finalization and dehydration. Prepare 50%hematin staining solution and 0.01%ammonia water or bluing reagent for post staining. Prewarm the target probes at 40 degrees Celsius for 10 minutes.
Bring all the RTU detection kit reagents except for DAB chromogen brown A and brown B to room temperature. To begin this assay de parize the baked tissue sections in xylene two times for five minutes each time with frequent agitation. Dehydrate in 100%Ethanol twice for three minutes each with frequent agitation.
Air dry for five minutes and then draw a hydrophobic barrier around the tissue section with the hydrophobic barrier pen. Start pretreatment of the tissue sections by incubating them with pretreat one solution for 10 minutes at room temperature. This will quench endogenous peroxidase activity.
Rinse in distilled water. Next retrieve RNA by incubating the tissue sections for 15 minutes with pre-treat, two solution maintained at a boiling temperature after 15 minutes, rinse twice in distilled water for protein digestion. Treat tissue sections with pre-treat three solution and incubate in the hybridization oven at 40 degrees Celsius for 30 minutes.
When the 30 minute incubation is done, rinse the tissue sections twice in distilled water. The next step is target probe hybridization using a pool of probes that detect the E six E seven mRNA of seven high risk HPV genotypes HPV 16, 18, 31, 33, 35, 52, and 58. Add the HPV probes, ubiquitin C probe and bacterial gene DAP B probe separately onto three adjacent tissue sections.
Hybridize at 40 degrees Celsius in the oven for two hours at the end of the two hour incubation. Rinse tissue sections in wash buffer twice for two minutes each time at room temperature for signal amplification. Begin by incubating tissue sections with the AMP one pre amplifier for 30 minutes at 40 degrees Celsius in the hybridization oven.
Rinse twice in wash buffer for two minutes each time at room temperature. Subsequent steps for signal amplification involve incubating the tissue sections with the following reagents at 40 degrees Celsius for the indicated amount of time with two rinses in wash buffer at room temperature. After each incubation, AMP two background reducer for 15 minutes.
AMP three amplifier for 30 minutes and AMP four labeled probe for 15 minutes. Next, incubate the tissue sections with amp five for 30 minutes at room temperature after the two rinses in wash buffer, incubate with amp six for 15 minutes at room temperature, followed by two more rinses in wash buffer. For signal detection, make a one one-to-one DAB mixture by mixing equal volumes of brown A and brown B.Add the solution to the slides and incubate for 10 minutes at room temperature.
Rinse twice in distilled water. Counter stain the tissue sections with a 50%hemat toin solution for two minutes at room temperature and then rinse with distilled water until slides are clear while tissues remain. Purple dip slides into 0.01%Ammonia water five times followed by five dips and distilled water dehydrate tissue sections by incubating for two minutes each in 70%ethanol, 100%ethanol and 100%ethanol, followed by xylene for five minutes.
Lastly, mount the slides with xylene based mounting media shown Here are example images from stained formal and fixed paraffin embedded sections of head and neck squamous cell Sonoma. In the HPV positive case, the HR HPV probes detected strong punctate signals specifically in the tumor cells. This is clearly visualized in the inset at 40 x magnification.
The positive control UBC probe detected numerous punctate cytoplasmic signals in both tumor cells and stromal cells. While the negative control DAP B probe demonstrated a clean background in the HPV negative case, both the HR HPV probe and the DAP B probe detected no signals, strong signals were detected by the UBC probe. The scoring for HPV status determination involves examining all three slides for each case.
The staining level on the negative control DAP B slide is used as the cutoff HPV positivity is defined by the presence of punctate, cytoplasmic and or nuclear staining above the signal on the DAP B slide. The RNA scope assay can be completed within eight hours if performed the correctly while performing the assay. It is critical to add sufficient amount of solution to cover the entire tissue area at each step.
It is also important to remove any residual buffer by flicking the slides between steps, yet keeping the slides white. The rescope technology can be applied for detection of essentially any RNA biomarkers in any cell or tissue of any species.
