With TRAP-seq, we can isolate the RNA from specific cell types, such as the hepatocytes repopulating in injured liver to analyze the gene expression changes occurring in those cells. It does not require any steps to remove unwanted cells from the tissue. The cell type-specific RNA is isolated by affinity purification from whole organ lysates.
This technique helps us determine how specific cells respond to injuries, which could help identify new strategies or drugs to fight liver disease. This could be used to identify how liver cells respond to different types of liver injuries. Currently, there are few options for severe acute liver injuries and this method will help to clue us in to new treatments.
This method originated in the brain. In the liver, we envisioned it could be used to examine dynamic changes in a variety of cell types, the hepatocytes, biliary duct cells, Kupffer cells, and endothelial cells to name a few. Demonstrating the procedure will be Amber Wang, a graduate student and my mentee.
To begin, resuspend magnetic beads by gentle pipetting. Collect the beads on a magnetic stand for more than one minute and remove the supernatant. Then, remove the microcentrifuge tube from the magnetic stand and add one milliliter of PBS, followed by pipetting up and down to wash the beads.
Place the tube back on the magnetic stand for more than one minute to collect the beads and remove the PBS. To prepare protein L-coated beads, add 16 microliters of biotinylated protein L to the resuspended and washed magnetic beads and add 1X PBS to make the final volume one milliliter, if using a 1.5-milliliter microcentrifuge tube, or 1.5 milliliters, if using a two-milliliter microcentrifuge tube. Incubate the magnetic beads with biotinylated protein L for 35 minutes at room temperature on a tube rotator.
Then, place the tube on the magnetic stand for more than one minute and remove the supernatant to collect the protein L-coated beads. Remove the microcentrifuge tube from the magnetic stand and add one milliliter of PBS with 3%BSA buffer, followed by gentle pipetting at least five times to wash the protein L-coated beads. Again, place the tube on the magnetic stand for more than one minute and remove the supernatant to collect the coated beads.
Repeat the BSA washing another four times. Next, add 50 micrograms of antibodies for GFP antibody 19C8 and GFP antibody 19F7 each into the protein L-coated beads, and incubate for one hour at four degrees Celsius on a tube rotator. To collect the affinity matrix, place the tube on the magnetic stand for more than one minute and remove the supernatant.
Take the tube away from the magnetic stand and add one milliliter of low-salt buffer. Gently pipet up-and-down to wash the affinity matrix and repeat the low-salt buffer washing another two times. Resuspend the beads in 200 microliters of low-salt buffer, to make 200 microliters of affinity matrix.
First, set up the tissue grinder so that the PTFE glass tubes can be placed on ice during homogenization. Fill four milliliters of cold lysis buffer into the PTFE glass tubes. Lay the liver on a Petri dish.
Use a knife to isolate 200 to 500 milligrams of liver pieces and move into the PTFE glass tubes. Place the remaining liver tissue into a pre-chilled microcentrifuge tube and flash freeze in liquid nitrogen. Homogenize the samples in a motor-driven homogenizer, starting at 300 rpm for at least five strokes to dissociate hepatocytes from the liver structure.
Lower the glass tube each time. Prevent aeration, which could cause protein denaturation, by keeping the pestle below the solution. Then, raise the speed to 900 rpm to fully homogenize the liver tissues for at least 12 full strokes.
Transfer the lysate into labeled and pre-chilled tubes with no more than one milliliter of lysate each. To start nuclear lysis, centrifuge the liver lysate at 2, 000 times g at four degrees Celsius for 10 minutes, then transfer the supernatant to a new pre-chilled microcentrifuge tube on ice. Add 1/9th of the supernatant volume of 10%NP-40 into the microcentrifuge tube on ice and mix the solution by gently inverting the tube.
Quickly spin down the microcentrifuge tubes with a mini centrifuge and add 1/9th of the sample volume of 10%DOC. Mix by gently inverting the microcentrifuge tubes and quickly spin down the microcentrifuge tubes and incubate on ice for five minutes. Centrifuge the nuclear lysate at 20, 000 times g at four degrees Celsius for 10 minutes and transfer the supernatant to new pre-chilled microcentrifuge tubes on ice.
For each tube, take out 1%of the total volume of the supernatant as a pre-immunoprecipitation control. Place the pre-immunoprecipitation controls on a tube rotator at four degrees Celsius overnight. Take extra care to gently resuspend the affinity matrix by pipetting, then add 200 microliters to each sample.
Incubate the lysates at four degrees Celsius overnight with gentle mixing on a tube rotator. To isolate RNA, quickly spin down the tubes containing the lysates in the mini centrifuge and collect the beads by placing on the magnetic rack for at least one minute. Collect the supernatant in additional microcentrifuge tubes.
Add one milliliter of fresh high-salt buffer to each tube, followed by gentle pipetting at least five times without introducing bubbles. Collect the beads on the magnetic stand on ice for over one minute and remove the supernatant. Repeat the washing steps another four times.
Then, remove the microcentrifuge tubes from the magnetic stand and place at room temperature for five minutes to warm up. Resuspend the beads in 100 microliters of lysis buffer with 0.7 microliters of beta-mercaptoethanol to release bound RNA from the affinity matrix. Vortex the tubes for at least five seconds at the highest speed and quickly spin down.
Then, incubate the tubes at room temperature for 10 minutes. Place the tubes on the magnetic stand for at least one minute, then collect the supernatant and proceed immediately to RNA cleanup according to the RNA purification protocol in the kit. To achieve maximum quality of the isolated RNA, perform all optional steps, including DNase digestion and all RNA elution steps, including the recommended preheating of the elution buffer to 60 degrees Celsius.
In this study, GFP-L10A fusion and Fah transgenes were co-delivered within a transposon-containing plasmid trap vector to livers by hydrodynamic injection. The removal of nitisinone induced a toxic liver injury. Immunofluorescence staining confirmed the coexpression of the FAH and GFP-L10A fusion protein and the repopulating hepatocytes after two weeks of liver repopulation.
The quiescent sample produced the highest yield of fusion protein, since all hepatocytes express GFP-L10A after AAV8-TBG-Cre injection. Conversely, barely any RNA was detectable in wild-type controls that did not have the GFP-L10A transgene, indicating the TRAP procedure is highly specific and has a low background. When TRAP was used on liver tissue undergoing repopulation with GFP-L10A transduced hepatocytes, abundant high-quality RNA was obtained.
In contrast, no RNA trace was detected via Bioanalyzer for the negative control sample. Gsta1 expression was induced by over tenfold in repopulating hepatocytes as compared to quiescent hepatocytes, while no threshold cycle values was detected with TRAP-isolated RNA from the wild type mouse due to the lack of input RNA. When homogenizing the tissue, make sure to move the pestle slowly to prevent aeration.
After isolating the RNA, high-throughput sequencing or qPCR can be performed to analyze gene expression. With TRAP-seq, we now have a method to specifically isolate RNA from repopulating hepatocytes and analyze the change in gene expression during liver repopulation. This allows us to identify genes that are altered during this process and potential therapeutic targets for the treatment of liver injury.
Cycloheximide and DTT, which are present in several buffers, are both toxic. Waste should be collected and discarded according to institutional guidelines.
