miRNA Expression Analyses in Prostate Cancer Clinical Tissues

The overall goal of this video is to describe a simplified workflow for micro RNA analyses in archived prostate cancer clinical specimens using a combination of quantitative real-time PCR and NC two hybridization with the implementation of locked nucleic acid-based probes. This is achieved by first isolating tissue from prostate cancer FFPE sections using laser capture microdissection, followed by a micro RNA extraction. Next two step R-T-P-C-R is used to quantify the micro RNA in the tissue and to localize the micro RNA in C two hybridization analysis is performed using digo oxygen and labeled LNA probes and AP conjugated antigen and antibody detected by BM purple.

The presented results show expression of a micro RNA in prostate cancer clinical tissues. Using these techniques Recently, microRNAs have emerged as promising alternate disease biomarkers for prostate cancer. However, microRNA expression analysis and prostate cancer clinical specimens is often challenging owing to confounding factors like heterogeneity of prostate tumors and factors like stromal contamination.

We have been working in the field of microRNA and non-coding RNA for the last 10 years. We have optimized the condition for micro RNA expression in different stage in greater prostate cancer. We believe that microRNA expression can be used as a diagnostic and prognostic biomarker because it correlates with the clinical outcome of prostate cancer.

We also believe that microRNA can be used for treatment of prostate cancer using nanoparticle technology. Accurate expression profiling in prostate carcinoma is often hindered by heterogeneity of the lesions. This is partly overcome by using the laser capture micro dissection or LCM to collect pure benign or malignant epithelial cells and reduce their mixture with the str cells.

LCM of archived FFP tissues, followed by microRNA expression profiling, has been found to be sensitive, reliable, and cost effective as a complementary technique optimized in C two hybridization offers a view of where the microRNAs are expressed Once tissues have been collected and made into 10 micron sections. Remove the paraffin in a fume hood, soak the slides in xylenes for 10 minutes two times, then rehydrate the tissues by moving them to a series of progressively aqueous alcohol baths. Next, apply a hemat toin stain for 30 seconds and rinse the slides in water.

Now rehydrated move the tissues through another graded alcohol series beginning with 70%Then bathe the sections in xylenes for five minutes and let them air dry. Take the dry slides to the laser capture microdissection instrument. Use pathologist marked slides to identify the prostate cancer foci and benign slash normal areas.

Follow the manufacturer's guidelines for performing laser capture microdissection. Set the beam width to 10 to 20 microns and pulse it with 70 to 90 milliwatts. Use infrared pulses to capture areas of interest onto laser capture micro dissection caps.

For each sample, add the contents of the cap to a collection tube and immediately process the cells for micro RNA extraction. Extract the total RNA using a commercial kit according to the manufacturer's instructions. Elute the RNA into 20 to 30 microliters of nuclease free water and proceed quickly.

Next, use a reverse transcriptase reaction kit to make CDNAs using micro RNA specific primers. Set up each reaction using 10 to 100 nanograms of total RNA. Once collected, dilute the CD NA by a factor of five to 10 for a concentration of about one to 20 nanograms.

Now set up the PCR reactions and triplicate from the CD NA using micro RNA assays with a fast universal master mix for the control. Use RNU 48 as a template to calculate the relative change in gene expression. Use the comparative CT method to begin pretreat the five micron thick tissue sections.

Fix the slides at 56 degrees celsius for an hour. Then depas the slides with two 15 minute xylene bath set room temperature following the xylenes, rehydrate the tissues through a graded ethanol series. Then soak the tissues in water for five minutes.

Next, fix the tissues with 4%PFA in PBS at room temperature for 20 minutes. Wash off the fixative with two five minute washes in PBS. Now load potent aase K at 10 micrograms per milliliter into prewarm proteinase K buffer upper, and incubate the tissues in the at 37 degrees Celsius for 10 minutes.

Following the treatment, rinse the slides with 0.2%glycine in PBS for 30 seconds and wash the slides in PBS at room temperature for five minutes. Then soak them in 4%PFA again for 15 minutes. Again, wash the fixative off with two five minute PBS baths.

The tissues are now ready to be hybridized. First, draw a hydrophobic circle around the tissue using a path pen so the solution in subsequent steps stays on the tissue section. Incubate the tissues in pre hybridization buffer for three to four hours at 55 degrees Celsius.

Do this in a chamber humidified by towels, soaked with 50%form amide, 50%five XSSC solution. Now prepare the diggen and labeled micro RNA specific probe and the control probe Plan on making 20 to 50 nanomolar dilution, but first dilute the probes with just 25 microliters of hybridization buffer less than needed for the final dilution. Next, heat the probes to 90 degrees Celsius for approximately four minutes.

Then put the probes on ice. Finally, further dilute the probes with ice cold hybridization buffer to their final concentrations of 20 to 50 nanomolar. Now apply 100 to 400 microliters of probe solution to each slide.

Then incubate them for 12 to 16 hours at the appropriate hybridization temperature. The next day, perform an SSC solution wash in several steps following the washes. Incubate the slides in blocking solution at room temperature for one to two hours following blocking.

Soak the tissues in one to 100 AP conjugated antio genin in PBS. Do this for one to four hours at room temperature or overnight at four degrees Celsius. Later, remove the excess antibody with three washes in PBS for 10 minutes per wash.

Follow with two five minute washes in a pee buffer at room temperature. Then incubate the slides in BM purple, AP substrate shielded from light at room temperature for one hour to 20 hours. The next day, remove the BM purple with a rinse in 0.1%between 20 in PBS.

Then wash them two times in water and mount them with aqueous mounting media using the described protocol of micro RNA extraction and R-T-P-C-R. microRNA 2 0 3 was examined in prostate cancer and normal adjacent tissues. The relative expression varied in a substantial fraction of tissues.

RNU 48 was used as a control. Prostate cancer tissues were either micro dissected under the microscope or were subjected to LCM and then analyzed the relative microRNA. 3 83 expression in tumor tissues was compared to matched to normal controls.

LCM prepared tissue detected micro RNA expression more reliably. In C two hybridization to microRNA 2 0 3 was performed in normal and bone metastatic prostate cancer tissue. U six's.

RNA detection was used as a control based on the intensity of staining. microRNA 2 0 3 expression was scored from one to four in cells of each tissue. The expression was thus found to be statistically lower in the bone metastatic tissue.

After watching this video, you should have a good understanding of how to assess microRNA expression in prostate cancer clinical specimen During the entire workflow. It is of utmost importance to maintain an RNA free environment. Also working with Paraform Hyde and saline can be extremely hazardous and precautions such as working in a fume hood and wearing personnel protective equipment should always be taken while performing this procedure.

Following this procedure, we perform statistical analysis to assess if micro expression patterns are correlated with the clinical pathological variables of the disease, which helps us to answer key questions like if the expression of a particular micro RNA has any associated prognostic or diagnostic potential, Our optimized microRNA in C two hybridization protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays. This protocol can also be applied to other tissue types with optimization.