The overall goal of this experiment is to identify useful molecular markers for sinonasal carcinomas and olfactory neuroblastomas in a simple and efficient way. This method can help answer key question in the field of olfactory neuroblastoma and sinonasal carcinoma, such as the identification of molecular markers to discriminate tumor subtype. The main advantage of this technique is that we can obtain result in less than six hour, or within a day for the hospital report.
Demonstrating the procedure of RNA extraction and real-time PCR will be Dr.Giorgia Millefanti, a PhD student from my lab. And demonstrating the procedure of immunohistochemistry will be Giovanni Micheloni, also a PhD student from my lab. To begin the procedure, collect and divide all the human FFPE samples into different sub-groups, according to the WHO classification of head and neck tumors.
Heat the samples slides in an oven at 60 degrees Celsius for 30 minutes. Next, rehydrate the three micrometer thick FFPE sections by briefly washing them in xylene for 10 minutes. Then, wash the slides in 100%95%85%and 75%alcohol serially for five minutes each wash.
Rinse the slides for five minutes in distilled water. Subsequently, block the endogenous activity by placing the slides in 3%aqueous hydrogen peroxide for 12 minutes. Afterward, perform antigen-retrieval by treating the slides with 10 millimolar citrate buffer, and microwaving for 10 minutes.
After 10 minutes, wash the sections in TBS buffer. Then, add 0.2%of Triton X, and incubate them overnight at four degrees Celsius, with Goat Anti-Human OTX2 Antibody, diluted at a 1 to 100 ratio. The next day, add biotinylated rabbit anti-goat secondary antibody, diluted at 1 to 200, and incubate the sections for one hour at room temperature.
Then, treat the samples with the ABC peroxidase complex. Afterward, develop the immunoreaction using diaminobenzene tetrahydrochloride, and counter stain the nuclei with Harris hematoxylin. Then, dehydrate the sections in a crescent alcohol scale, and clarify them in a clearing substance of turpine origin.
Next, mount the sections on a slide with mounting medium. After RNA extraction and reverse transcription, perform quantitative real time PCR analysis with probe-based technology and a thermal cycler. Prepare the PCR reaction mix using 12.5 microliters of probe-based master mix, 1.25 microliters of each OTX1, OTX2, and ACTB probes, 50 nanograms of CDNA, and nuclease-free water up to 25 microliters of total volume.
Perform all the reactions in triplicate using ACTB gene as the endogenous control to normalize gene expression levels. Next, centrifuge the plate at 1109 times gravity for three minutes. Then store the plate protected from light at four degrees Celsius, until the experiment.
To perform real time PCR, place the samples in the machine. Then, set the thermal cycler profile with an initial hot start cycle at 50 degrees Celsius for two minutes, and 95 degrees Celsius for 10 minutes, followed by 40 cycles at 95 degrees Celsius for 15 seconds and a final cycle at 60 degrees Celsius for one minute. For gene expression level analysis, normalize the gene expression levels through the comparative cycle threshold method using ACTB gene as the endogenous control.
Evaluate the gene expression levels using the comparative cycle threshold method, and plot the results. Then, perform statistical analysis using student's t-test, considering statistically significant results with p<0.05. In the normal mucosa, strong and homogenous nuclear reactivity for OTX genes both in ciliated pseudostratified respiratory type epithelium and in the submucosal glandular cells were observed.
Nuclear expression for OTX1 was found in all non-intestinal type adenocarcinoma samples, while little or absent immunoreactivity was highlighted in intestinal type adenocarcinomas. Intense immunoreactivity was present in all olfactory neuroblastomas. Among all poorly differentiated neuro-endocrine carcinomas, OTX expression varied in intensity and percentage of positive cells.
While attempting this procedure, it is important to remember to perform every single step of the RNA extraction in sterile conditions using also sterile equipment. In parallel with this procedure, other methods like x-ray analysis, endoscopic exam of nasal cavity, CT scan, magnetic resonance imaging, and biopsy, can be performed in order to confirm the results obtained from our technique. After its development, this technique paved the way for researchers in the field of oncology to explore this disease in cell models.
After watching this video, you should have a good understanding of how to identify molecular markers to discriminate tumor subtypes using the immunohistochemistry and real time PCR. Don't forget that working with these reagents, instrumentation, and samples can be extremely hazardous and precautions such as DPI should always be taken when performing these procedure.
