Hi, I'm Bin Ren, I'm an associate professor of surgery and biomedical engineering at University of Alabama the Birmingham School of Medicine. Our laboratory's interested on endothelial cell biology function angiogenesis acuogenesis in the context of these common cardiovascular diseases and cancers. One of our research focuses is on epigenetic and transcription regulation of endothelial cell transdifferentiation and angiogenesis.
To understand the transcriptional mechanisms of angiogenesis, we have established transgenic engineered mouse lines, in which we have not only deleted a specific genes associated with angiogenesis in the endothelial cells, but an enhanced GFP probe will be genetically tagged onto a ribosome of native endothelial cells. In this way, we can isolate and purify loops of associated messenger RNA from endothelial cells in different tissue organs directly in transgenic animals. We can then use the purified messenger RNA for downstream analysis of transcripts and the transcriptome.
By RNA sequencing and the real time chill PCR. In this video, my students Patrick Moran and Jordan Palmer and the research technicians Nicholas Barnekow and Rong Yuan will show you an approach genotyping transgenic mice and the aspirating messenger RNA directly from the endothelial in the transgenic animals. Genotyping is an important preliminary in this protocol to ensure that the ice have the GFP tag on the ribosomes of endothelial cells.
Since all downstream RNAi isolation steps rely on the presence of GFP tagged ribosomes, it is essential to determine the genotype prior to beginning the experiment. Prior to beginning the RNA isolation, several preparatory steps must be completed. These include buffer preparation and binding the anti-GFP antibody to the protein G dina beads.
The buffers can last up to one month at four degrees Celsius and the antibody-bound beads can last up to one week at four degrees Celsius. So take these limitations into account when planing your experiment. Using a molar driven homogenizer is an excellent way to increase RNA yield.
Be sure to use lower frequency settings and durations in order to reduce RNA degradation. As mentioned earlier, genotyping is an important preliminary step in the trial protocol. Ensuring that mice whose RNA is to be isolated using the TRAP technique are positive for the TRAP gene, at least heterozygotes, but ideally homozygous for the TRAP gene is an important step because ensuring the GFP tag is on the ribosome is essential in order to pull down using the antigen antibody reaction and the anti-GFP beads you will have prepared prior to beginning the experiment.
Isolate the desired tissues from the mouse and place it immediately into an ice cold PBS with 100 microgram per milliliter cyclohexamide solution. This ensures that translation will be stopped and that the transcriptome will be an accurate depiction of the mouse prior to euthanasia. Use a motor driven homogenizer to homogenize the tissue into a cell suspension.
Ideal timing and frequency settings will have to be determined by the individual lab based on the particular motor driven homogenizer you will be using. Using too high of a frequency setting can lead to RNA degradation and disrupt the rest of the protocol. Suspend the cell pellet within the lysis buffer you should have prepared prior to beginning the experiment.
Be sure to homogenize this mixture further in order to ensure adequate homogenization prior to proceeding with the rest of the protocol. Centrifuge the homogenate for 10 minutes at 2, 000 times G in a four degrees Celsius centrifuge. This will pellet the nuclei and large cellular debris which may interfere with the downstream immunoprecipitation reaction.
Add DHPC and CA-630 lipids to the supernatant from the prior centrifugalization step. These lipids will help separate the protein phases out from the other intracellular components. Place the suspension on ice for five minutes and allow it to sit.
Centrifuge the lysate for 10 minutes at 13, 000 times G to pellet any soluble material. Keep 15%of the clear lysate for future steps. Add the anti-GFP antibody bound beads to the cell lysate supernatant and incubate the mixture at four degrees Celsius with end over end rotation for 30 minutes.
This is where the anti-GFP antibodies will bind the GFP tagged ribosomes, allowing us to further isolate the RNA from these ribosomes. Collect the beads on your magnetic rack and wash the beads five times using your high salt polysome wash buffer that you will have prepared prior to beginning the isolation. Immediately proceed to the next step in which you will place the beads in the RLT buffer.
Centrifuge the lysate for three minutes at full speed and carefully remove the supernatant and transfer it to a new microfuge tube. Add one volume of 70%ethanol to the cleared lysate and mix immediately by pipetting. Proceed immediately to the next step in which you will transfer up to 700 microliters of the sample, including any precipitate that may have formed, to a spin column placed in a two milliliter collection tube.
Centrifuge the lysate for 15 seconds at greater than 8, 000 times G to wash the spin column membrane. Discard the flow through and proceed to the next step. Add 350 microliters of buffer RW1 to the spin column.
Centrifuge for 15 seconds at greater than 8, 000 times G to wash the spin column membrane and discard the flow through. Repeat this step using another 350 microliters of buffer RW1 before proceeding to the next step. Add 500 microliters of buffer RPE to the spin column.
Centrifuge for 15 seconds at greater than 8, 000 times G to wash the spin column membrane. Discard the flow through and proceed to repeat this step by adding another 500 microliters of buffer RPE to the spin column. This time, centrifuge for two minutes at greater than 8.000 times G to wash the spin column membrane.
Discard the flow through and place the spin column in a new two milliliter collection tube and proceed to centrifuge at full speed for one minute to remove any residual buffer that may be present on the spin column. Discard the flow through and place the spin column in a new 1.5 milliliter collection tube. Proceed to add 30 to 50 microliters of RNase free water directly to the spin column membrane.
Centrifuge for one minute at greater than 8, 000 times G to elute the RNA from the spin column membrane. At this point, your RNA is dissolved in RNase free water and may be stored at minus 80 degrees Celsius for up to one year. Proceed to quantify and check the purity of the RNA you have isolated using a spectrophotometer.
You are looking for a peak at 260 nanometers, which is nucleic acid, including RNA, absorbs maximally. Quantity is depicted in the lower right portion of the image in nanograms per microliter. Quality can be inferred by the 260 230 ratio which is shown one item above the quantity in the lower right portion of the screen.
Our approach showed low RNA yields, especially when we purified mRNA from the heart tissues or from previously frozen tissues. We thus need to optomize the conditions to increase yields. However, we did observe in EC specific CD-36 deficient mice, the level of ephrin B2 and DLL4 were significantly increased in both lung and heart endothelia, when compared with the control.
These results were consistent with our previous in vitro studies, which suggests that the RNA quality is sufficient for downstream analysis. The yield was low due to the stringent conditions in our workspace. To overcome this limitation, it is critical to set up an RNase free work zone and decontaminate work surfaces and equipment that may get contaminated with RNase and change gloves frequently in order to extract quality RNA and increase yields.
It is also critical to find suitable concentrations of GFP antibodies in the affinity matrix and use appropriate concentrations of RNase inhibitor in the tissue lysis buffer and use RNase free plastic ware and reagents for RNA extraction from endothelial ribosomes of the targeted tissues.
