The overall goal of this procedure is to define microRNAs that estrogen receptors may regulate. This is accomplished by first preparing the cells for maximal estrogen response through culture to an optimal confluence and depletion of estrogenic activity. The second step is to induce transcriptional activity of the estrogen receptors by treatment with estradiol for the required number of time points.
Appropriate controls are included for comparisons. Next, the RNA, including the micro RNA population is extracted and converted to CD NA known estrogen regulated. Target genes are then measured using QPCR to confirm that the treatment protocol was successful.
The final step is to determine the genome wide micro RNA expression pattern using large scale screening technologies. Ultimately, confirmatory analysis using QPCR is performed. This method can help answer key questions in the breast cancer field, such as whether and how estrogen affects micro RNA expression.
Though this method can provide insight into breast cancer, it can also be applied to other systems such as other cancers or cell types that express either estrogen receptor alpha or bitter. The MC seven cells used in this study are prepared five days prior to treatment in a sterile hood. Remove media from the T 75 flask using a sterile pasture pipette connected to a vacuum.
Gently wash the attached cells twice with PBS and detach the cells by trypsin. Count the cells using a cell counter according to the manufacturer's protocol. Label six 100 milliliter plates and add 10 milliliters of media to each plate.
Plate about 2 million cells per plate and distribute the cells by gently swirling the plate. Incubate the cells for 24 to 48 hours at 37 degrees Celsius and 5%CO2 until the cells are approximately 80%confluent. Two to four days later, take the cells out of the 37 degrees Celsius incubator and observe under a microscope to ensure that they are approximately 80%confluent.
Cell con fluency is an important factor because it influences cell to cell contact and behaviors such as cell to cell signaling and proliferation. Wash the cells twice with PBS and then add the appropriate media with 5%FBS that has been filtered in order to remove estrogenic compounds. Dexter coated charcoal treated FBS or D-C-C-F-B-S incubate cells for 24 hours at 37 degrees Celsius and 5%CO2 on the following day.
Wash the cells twice with PBS and add the appropriate 0.5%D-C-C-F-B-S media incubate the cells for an additional 48 hours on the day of treatment. Prepare the reagents for cell treatment in a 50 milliliter conical tube. First, add 30 milliliters of the appropriate 0.5%D-C-C-F-B-S media, followed by three microliters of a 0.1 millimolar E two ligand stock wash cells twice with PBS.
Be sure to remove as much of the PBS as possible. Mix the contents of each tube gently and add 10 milliliters to the cells in each plate. There should be one ligand per plate.
Incubate the cells at 37 degrees Celsius and 5%CO2 for a chosen time period between zero and 72 hours. Replace the media as necessary after completion of treatment for the desired period of time. Remove the plates from the incubator and take them to a fume hood.
Since this procedure uses a toxic guana dium thiocyanate phenol solution, use appropriate personal protective equipment, wash the cells twice with PBS. Then add one to two milliliters of guad dium thiocyanate phenol solution to the cells in each plate. Ensure that the volume of cells is no more than 10%of the volume of guad dium thiocyanate phenol solution and that the plate is covered with the solution.
Let the cells sit for one minute. Next, use a rubber scraper to scrape the cells and transfer the cells to a micro centrifuge. Tube cells can be stored in this solution at minus 80 degrees Celsius for weeks.
Extracting RNA of high quality is very important for the success of this procedure. Therefore, the proceeding steps must be performed under RNA. Free conditions To extract total RNA from each treatment vortex a cell mixture for one minute and spin quickly.
Add 200 microliters of chloroform per one milliliter of guad dium thiocyanate phenol solution. Vortex for 15 seconds and incubate for two to three minutes at room temperature centrifuge at 11, 000 RPM and four degrees Celsius for 15 minutes. Transfer the upper aqueous phase to a new micro centrifuge tube.
Add 1.5 volumes of 100%ethanol and mix by pipetting. Transfer the solution to a spin column and purify the RNA by micro RNA spin column purification, followed by DN a's 1D NA degradation according to the manufacturer's protocol. Subsequently, the RNA is alluded in 60 microliters of RNAs free water aliquot, one to two microliters of RNA for quantification and quality analysis, and store the rest of the RNA at minus 80 degrees Celsius.
After confirming that treatment of cells resulted in regulation of known estrogen responsive targets such as mRNA micro RNA profiling is performed using both the micropower flow microfluidic, microarrays and dual labeled probes, low density arrays or D-L-P-L-D-A to perform micro RNA profiling using D-L-P-L-D-A, remove the D-L-P-L-D-A plates from the refrigerator and let them sit at room temperature while running the reverse transcription reaction to generate CDNA from the RNA samples. When the D-L-P-L-D-A plates have reached room temperature load 100 microliters of CDNA mixed with the dual label probes. PCR master mix into each of the eight fill ports of a plate.
Centrifuge the array plates. Briefly open the Q-R-T-P-C-R system and check that the block is for a 384 well plate. Set up the run.
Using the SDS software, select relative quantification. Select the well number and array type and enter sample descriptions to define the wells. Run the array using the default thermal cycling conditions.
When the run is finished, save the results, export the results to Microsoft Excel and save for further analysis. One method to confirm the micro RNA regulations is by cyanide D-Q-P-C-R analysis. The first step is to design primers the sequence of the mature micro RNA, which equals the forward primer can be obtained from mere based.org.
Convert all the UES to ts next, prepare the CD NA place one microgram of total RNA and a 1.5 milliliter centrifuge tube. Include a negative control with water instead of total RNA. Follow instructions for poly A tailing and first strand CD nna.
Synthesis of micro RNA from a micro RNA first strand cDNA synthesis and Q-R-T-P-C-R protocol. Dilute the resulting CD NA one to 10 with purified water and store at minus 20 degrees Celsius. Obtain 1 96 well reaction plate.
Prepare A-Q-P-C-R master mix. For each micro R-N-A-Q-P-C-R master, well add two picaMoles of each of the specific forward primer and the universal primer. Five microliters of two X-Q-P-C-R master mix and purified water.
Aliquot the mix into each. Well finally add 16 nanograms of poly a CD NA for each sample and include negative controls. The final reaction volume should be 10 microliters per Well Ensure that there are triplicate wells for each sample for each micro RNA for technical replicates as well as for negative controls.
In addition, include one or several reference genes, usually U six s nrp, RNA and or a micro RNA that is not changed on the Q-R-T-P-C-R system software. Assign the reporter and target Enter the reaction volume. Select the comparative threshold cycle method and define the sample wells.
Run plates using the default settings for the run. Make sure a melting curve analysis is performed for all cyan die runs at the completion of the run. Save the results and export all the data, including the CT values to Microsoft Excel.
Efficient extraction of high quality RNA from the samples is an important factor for the success of this study. After extraction, RNA concentrations are measured with a spectrophotometer as shown in this representative result. Each peak illustrated in the top panel represents a sample and a successful extraction process yielding pure RNA.
In contrast, a poorly extracted RNA illustrated in the bottom panel shows no clear absorption peak at 260 nanometers. For RNA quality control, RNA integrity was measured and the results compared with an RNA ladder. The 18 s and 28 S-R-R-N-A should have peaks in a good quality RNA sample as shown here.
A degraded RNA sample would have less clear peaks and a reduced relative amount of the 18 s and 28 S-R-R-N-A as shown here. Analysis of micro NA population can be performed separately. The micro RNA microarray results from a comparison between vehicle and E two treated samples are graphically represented by this heat map.
Mere a, mere B and mere C are upregulated in the E two treated samples as indicated in red, while mere D, mere e, mere F and mere G are downregulated in the E two treated sample as indicated in green. The micro RNA selection depends on their significance and expression. A P value less than 0.05 is usually considered significant and should be selected for further validation with QPCR.
The dual labeled probes low density arrays use A-Q-P-C-R based method to screen for regulated microRNAs. In a 384 well format amplification plots for micro RNA from the D-L-P-L-D-A results are shown here. Amplification of expressed microRNAs is indicated by increasing RN values.
Validations can be performed using two QPCR detection methods to prevent method bias and to allow for thorough confirmation and investigation of micro NA regulations. The first method uses cyanide dye detection to analyze the melting curve of a micro RNA as a control for amplification.Specificity. Amplification of the intended fragment will result in a clearly defined uniform peak as shown in the left panel.
Multiple peaks would be observed if the primer is nonspecific or if specific amounts of primer dimer are formed as illustrated by the right panel. If this occurs, the data cannot be used for expression analysis. The second method, dual labeled probes, QPCR analysis is more specific as only amplification of the target micro RNA is detected amplification plots of each target.
Micro RNA can be observed as shown here for both QPCR assays. A comparison to a reference gene is required to determine the relative expression of genes between two samples. An example of a suitable reference gene in these breast cancer cell lines is A-R-H-G-D-I-A-A RO GDP dissociation inhibitor.
As observed in this figure, the mRNA level of A-R-H-G-D-I-A is not changed between the vehicle and the ligand treated samples. A reference gene that is widely accepted for micro RNA analysis is U six snrp, RNA. The approximately 110 nucleotide long non-coding small nuclear RNA that functions in nuclear prem.
A splicing as observed in this figure U six expression levels are about the same across the samples shown, thus allowing for calculations of the variable levels of micro RNA. This final figure shows a graphical representation of micro R-N-A-Q-P-C-R results from vehicle and E two treated cells, comparing relative expression levels of a specific micro RNA mere 135 A over a time course. This micro RNA was detected as non-regulated up to 24 hours after E two treatment, but significant regulation was detected 72 hours after treatment Following this procedure.
Other methods like chromatin immunoprecipitation of the SDR N receptor can be performed in order to answer additional questions like whether the microRNA is directly regulated by the receptor through binding to CIS regulatory chromatin regions. After watching this video, you should have a good understanding of how to analyze the estrogen mediated regulation of microRNAs in a robust fashion. It is very important to perform the various controls and replication of the steps as outlined in this protocol to ascertain that you detect true regulations and not biological or technical variations.
