The overall goal of the following experiment is to quantify the expression levels of micro RNA molecules in prostate cancer and normal prostate tissue using quantitative real-time PCR. This is achieved by first extracting total RNA from prostate tissues. That includes Mr NA micro RNA, and other small RNAs.
Next, the RNA is reversed transcribed to generate CDNA. Then cyber green based real-time PCR is carried out using the CD NA as a template to amplify and quantitate micro RNA expression in real time. Results are obtained that show relative expression of microRNAs in various prostate samples based on quantitative realtime PCR.
The main advantage of this technique over other methods is that it offers great sensitivity, accuracy, reproducibility, and it is fast and cost effective. This method can be used to answer key questions in the field of cancer biology as microRNA expression is often correlated with genes that are heavily involved in the cancer process. We can therefore use QPCR to assess the expression of microRNAs and see how this correlates with clinical variables.
These biomarkers may potentially be used as both diagnostic and prognostic tools. They may help us diagnose prostate cancer as well as help us determine which prostate cancers are aggressive. In addition, there is potential for these biomarkers to be therapeutic agents.
Not only can this method provide insight into prostate tumor genesis, it may also be applied to other cancers and diseases. Collect prostate samples at the time of ectomy and orient the specimen using anatomic landmarks. Paint the prostate and the seminal vesicles as follows, right side green, left side blue.
Take a random transverse midsection of the tissue perpendicular to the rectal surface, freeze the sample and store it negative 80 degrees Celsius. Take a photocopy and orient the slices of specimens and divide them into four equal parts. Consult a pathologist to determine the tumor versus normal areas on the corresponding image.
Use the marked areas as a guide to identify tumor tissue for extracting RNA To isolate RNA. Begin by placing frozen prostate samples on dry ice and referring to the delineated photocopy. Cut out a small portion of the prostate tumor.
Homogenize the prostate tumor tissue in one milliliter of triol reagent. Incubate the homogenized samples for five minutes. At room temperature, add 0.2 milliliters of chloroform to the samples and shake vigorously for 15 seconds.
Incubate the samples for three minutes at room temperature, then centrifuge at 12, 000 times G for 15 minutes at four degrees Celsius. Transfer the colorless upper aqueous phase to fresh tubes and add 0.5 milliliters of isopropyl alcohol. Incubate samples for 10 minutes at room temperature, then centrifuge 12, 000 times G for 10 minutes at four degrees Celsius.
Carefully aspirate the supernatant without disturbing the pellet containing the RNA. Add one milliliter of 75%ethanol to the RNA pellet vortex, the sample and sediment by centrifugation for five minutes at 7, 500 times G at four degrees Celsius. Again, aspirate the SNAT and dry the RNA pellet for five to 10 minutes, making sure it is not completely dry.
Red, dissolve the pellet in nuclease free water and use a spectrophotometer to measure the concentration of the RNA. Use a bioanalyzer to check the quality and integrity of the RNA samples. Reverse transcribe the RNA into CDNA using a reverse transcription kit according to the manufacturer's instructions.
To generate a standard curve, prepare a series of dilution of the original CD NA from a sample that is known to have a substantial expression of the gene of interest. Perform real-time PCR by preparing a master mix based on a 20 microliter reaction volume of two x quant. Detect cyber green PCR master mix 10 x microscript universal primer, 10 x microscript primer assay and RN a's free water.
Include two sets of primers, one for the target gene and another for the reference gene in this case, RNU six B.Pipette two microliters of each CDNA dilution into individual light cycler capillary tubes, and add 18 microliters of the master mix. Use a capillary adapter to centrifuge. Place the capillaries into a capillary based realtime cycler and run the PCR program as follows, 95 degrees Celsius for 15 minutes, followed by 50 cycles of 94 degrees Celsius for 15 seconds.
55 degrees Celsius for 30 seconds and 70 degrees Celsius. For 30 seconds, select a sample to be the calibrator and set its normalized target amount to one. Compare the relative expression of the micro RNA in all the other samples to the calibrator to generate amplification curves.
For the PCR reactions, use the molecular biochemicals light cycler software version 3.5 in the quantification tab. Quantify the reactions and export the data to a text file. Import the data to the REL quant analysis software to generate quantification results.
Import separate files for target gene, reference gene and standard curve data. Specify the position of the calibrator for both target and reference gene, as well as the positions of the samples. Data is expressed as the target to reference ratio of different samples divided by the target to reference ratio of the calibrator.
Use the standard curve previously generated for a particular micro RNA and housekeeping gene as a reference standard for extrapolating quantitative data for micro RNA. Targets of unknown concentrations. Analyze three samples as a group and calculate the mean concentrations and standard deviations of the triplicate.
If one of the triplicates is inconsistent with the rest of the set, the program will exclude it. Shown here is an example of A-Q-P-C-R analysis of prostate samples. The graphs showing the expression levels of the reference gene U six begin exponential amplification at about cycle 20 while expression of the target gene, MIR 98 showed delayed amplification around cycle 25.
The data from this experiment were exported as a text file and analyzed by requ analysis software positions of the capillaries containing the calibrator and samples are specified. Once mastered, this technique can be done in one day when performed properly. Reliability of this technique depends on the quality of RNA.
Therefore, it's important to take extra care in handling RNA because it can degrade very easily.
