Chill 1.5-milliliter RNase-free conical tubes, a mortar and pestle and a spatula in liquid nitrogen according to the text protocol. Then, isolate the PRF from the kidney by cutting and removing the renal capsule.Īfter isolating the right PRF, transfer the sample to the aluminum foil with the tissue from the left side and use liquid nitrogen to freeze the samples. Now, isolate the PRF from the back muscular wall and cut the ureter, the renal artery and vein which separates the PRF and kidney from the rest of the body. This can be tedious as they are just a bit pinker than the adipose tissue. Isolate and remove the adrenal glands before collecting the PRF. The adipose tissue seen here surrounding the kidney and the adrenal glands is the perirenal fat, or PRF. Starting with the left-hand side, expose the kidneys by moving the gut away from the abdominal cavity to the right-hand side. Then, freeze the sample in liquid nitrogen. Detach the left and right fat pads from the epididymis, the testes and the ductus deferens and transfer it to the pre-labeled aluminum foil. The fat around the reproductive organs is the perigonadal fat, or PGF. Then, make an incision in the abdominal muscles from the genitals towards the back of the mouse on both sides. To get access to the visceral adipose tissue, cut the abdominal muscle along the linea alba. Then, pull both the left and right collected fat pads in the aluminum foil and use liquid nitrogen to freeze the sample. Repeat the dissection for the left side of the animal.
#Processing pmouse skin
Using surgical scissors positioned against the skin as flat as possible, carry out blunt dissection of the abdominal subcutaneous fat, or SCF, then transfer it to a precut piece of aluminum foil. Stretch one corner of the opened skin and use a 22-gauge needle to pin it down on the pad.
After euthanizing a mouse, cut the skin over the linea alba according to the text protocol, then make two incisions of approximately two centimeters from the middle of the ribcage towards the right arm and close to the genital organs above the right hind limb. The main advantage of this technique is that it allows for the isolation of high amounts of good-quality RNA from adipose tissue which typically has low RNA content.ĭemonstrating the procedure will be Emilie Pepin, a research assistant from my laboratory.
This method can answer key questions in the fields of metabolism, biochemistry and molecular biology that, for example, pertain to gene expression in adipose tissue from treated or genetically-modified mice. Published by Cold Spring Harbor Laboratory Press.The overall goal of this procedure is to ensure adequate sample collection of white adipose tissue depots from different mice, and to isolate a high amount of good-quality RNA from the tissue.
#Processing pmouse how to
This yields some of the first genome-wide insights into DSB resection and repair in a mammalian genome and offers a tantalizing glimpse of how to quantitatively dissect this difficult to study, yet integral, nuclear process.ĪTM DNA double-strand breaks EXO1 PRDM9 chromatin meiosis recombination resection. 806-818) capture some of the first details of resection and DSB repair intermediates in mouse meiosis using a method that maps blunt-ended DNA after ssDNA digestion. In this issue of Genes & Development, Yamada and colleagues (pp. The nucleolytic resection of DSB-adjacent DNA is a key step in meiotic DSB repair, but this process has remained understudied. To generate a crossover, hundreds of DNA double-strand breaks (DSBs) are introduced in the genome of each meiotic cell by the SPO11 protein. The exchange of genetic information between parental chromosomes in meiosis is an integral process for the creation of gametes.
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