The overall goal of this experiment is to understand the direct metabolic regulation of ER Alpha in breast cancer, using genome-wide transcriptome, systrome, and metabolome data. This method can help answer key questions in the nucleoreceptor and cancer biology fields, such as the role of certain transcription factors in regulating metabolism to contribute to cell aggressiveness. The main advantage of this technique is that it gives a global picture of gene and resulting metabolic regulation nuance.
Demonstrating the procedure will be Yiru Chen, a postdoctoral fellow from my laboratory. After culturing MCF7 cells according to the text protocol, harvest the cells by adding one milliliter of RNA isolation reagent to each well, and incubate at room temperature for five minutes. Use a pipette to collect the cell lysate and transfer it into 1.5 milliliter tubes.
To isolate the RNA, add 200 microliters of chloroform to one milliliter of cell lysate, and vortex for 10 seconds. After incubating at room temperature for five minutes, centrifuge the samples at 16, 000 x g and four degrees Celsius for 15 minutes. Following centrifugation, carefully transfer the aqueous top phase to a new tube.
Add 500 microliters of 100%isopropanol to the aqueous phase, and mix by inverting. Then, incubate at negative 20 degrees Celsius overnight. The following morning, centrifuge the samples at 1600 x g and four degrees Celsius for 10 minutes.
Discard the supernatant, and add 750 microliters RNase-free 70%ethanol, and briefly vortex. Centrifuge the samples at 6, 000 x g and four degrees Celsius for five minutes. Then, remove the supernatant, and centrifuge the samples at 16, 000 x g and four degrees Celsius for one minute.
With a gel loading tip, remove the remaining ethanol by pipetting. And set the tubes upside-down to air dry for 10 minutes. Then, add 20 to 50 microliters of DEPC water to dissolve the pellet, and purify and quantify the RNA according to the text protocol.
Once MCF7 cells have been cultured according to the text protocol, add 250 microliters of 16%formaldehyde into five milliliters of medium to the cells to cross-link chromatin and incubate at room temperature for 15 minutes. Then use five milliliters of ice-cold 1X to wash the cells and stop the cross-linking reaction. Harvest the cells in 250 microliters of cell lysis buffer per plate.
Then, to fragment the chromatin to the required sizes, sonicate the cells eight times for 30 seconds each at an amp of 30. Cool each sample on ice after each 30 second sonication. Next, after centrifuging the samples at 16, 000 x g and four degrees Celsius for 10 minutes, carefully pipette the supernatant without disturbing the pellet at the bottom.
Apply a five microliter aliquot of the supernatant to a 1%agarose gel, and carry out electrophoresis to determine the DNA size. The ideal fragment length should be between 200 and 500 base pairs. To carry out immunoprecipitation of the chromatin, save 25 microliters of the cell lysate as input.
Then, in a 50 milliliter tube, mix four milliliters of the cell lysate with 20 milliliters of IP buffer, ER Alpha antibody, and protein A and protein G-coded magnetic beads. Incubate the mixture by rotation at four degrees Celsius overnight, to allow the formation of chromatin-antibody complexes. After using wash buffers to wash the beads, according to the text protocol, add 500 microliters of elution buffer to the beads, and elute the DNA into four tubes.
For the DNA input samples, elute into 125 microliters of elution buffer. Next, place the tubes in a heating block and cover them with foil, and place a weight on top to prevent them from opening. Incubate the tubes at 65 degrees Celsius overnight.
To isolate the DNA from the pulldown samples using a ChIP DNA isolation kit, cool the tubes at room temperature for five minutes. In the meantime, warm up the elution buffer to 65 degrees Celsius. Add 625 microliters of binding buffer to each tube.
If a precipitant forms, add another 250 microliters of binding buffer until the solution is clear. Load the sample onto the column and centrifuge at 16, 000 x g for 30 seconds. Then use 250 microliters of wash buffer to wash the column twice.
With 15 microliters of elution buffer, elute the DNA. Combine the DNA from the four tubes of the same pulldown to arrive at 55 microliters of DNA. Measure DNA concentration and carry out QPCR according to the text protocol.
Using MCF7 cells cultured according to the text protocol, add five milliliters of ice-cold 1X PBS to the plate. Then tilt the plate several times before removing the PBS. Repeat two more times, removing as much PBS as possible after the last wash.
Add 750 microliters of pre-chilled acetone to each plate, and scrape the cells. Then, combine the cells from two plates into a two milliliter tube on ice. To count the cells for normalization, for each treatment, use two extra plates for cell quantification.
Then, to detach the cells from the plates, add two milliliters of HE buffer and incubate for five minutes. Mix the cells thoroughly by pipetting in the HE buffer. Then, use 20 microliters of the cell solution in a hemocytometer to count the cell number under a microscope.
Store the samples at negative 80 degrees Celsius before using gas chromatography mass spectrometry analysis to identify and quantify the metabolites. Perform integrative analysis according to the text protocol. This figure shows the integrity and overall quality of RNA samples purified for an RNA-Seq experiment.
The analysis shows all samples have sharp and clean bands of 18S and 28S RRNAs, indicating the integrity and purity of the samples. An RNA Integrity Number, or RIN, of 10, indicates intact RNA. Six represents partially degraded RNA, and a three indicates a sample that is strongly degraded.
In these experiments, all RNA samples received RIN values between 9.6 and 9.9. Carrying out ultra high throughput sequencing resulted in approximately 18 million high-quality sequencing reads per sample. A representative fast QC report, which analyzes the overall quality of the raw data, is shown here.
For each read, the quality score was 32 to 34 for the first five base pairs, and 36 to 38 from six to 100 base pairs. Among 17, 990, 863 reads generated from this sample, most of them had a quality score higher than 34. This figure shows an overview of about 100 metabolites represented by peaks that show changes in MCF7 cells due to estradiol treatment.
The top 10 metabolites with significant changes from E2 treatment are presented in this table. And this table lists the pathways that were up-and down-regulated upon E2 treatment. Once mastered, this technique can be done in a week if it is performed properly.
While attempting this procedure, it's important to remember to pay attention to the health of the cells, and quality of the samples that will be analyzed. Following this procedure, other methods like exome sequencing or GRO-Seq that rely on sequencing data can be performed in order to answer additional questions like the identity of additional MRNAs, enhancer MRNAs, or long non-coding RNAs whose transcription are induced by treatments. After its development, this technique paved the way for researchers in the field of nuclear receptors to explore metabolic regulation by nuclear receptors in cell culture systems or in mice.
After watching this video, you should have a good understanding of how to analyze and integrate data from multiple Omics approaches. Don't forget that working with Trizol, formaldehyde and methanol can be extremely hazardous and precautions such as wearing personal protection equipment such as gloves and lab coats should always be worn while performing this procedure.
