Mouse ribopure blood rna isolation kit

Blood sample was diluted with equal amount of sterile phosphate-buffered saline (PBS) solution (GE Healthcare Life Sciences). One milliliter of Ficoll-Paque (GE Healthcare Life Sciences) was inserted into a 2 ml Eppendorf centrifuge tube. Diluted blood sample was layered carefully onto the Ficoll-Paque. Then sample was centrifuged at 400 gav for 30 min at 18 °C. After removing an upper layer, a lymphocyte layer was transferred into a new 2 ml Eppendorf tube, filled with PBS solution, mixed and afresh centrifuged at 100 gav for 10 min at 18 °C. Then the supernatant was discarded carefully not to disrupt the pellet of the peripheral blood mononuclear cells (PBMC). Subsequently, PBMCs were washed again with PBS solution (centrifugation repeated under the same conditions). Afterwards 800 µl of lysis solution and 50 µl of sodium acetate (Mouse RiboPure™-Blood RNA Isolation Kit; Invitrogen; Thermo Fisher Scientific) was added, the cells were suspended by manual pipetting, then the samples were placed immediately at −80 °C. Total RNA was isolated with the use of Mouse RiboPure™-Blood RNA Isolation Kit (Invitrogen; Thermo Fisher Scientific) according to the manufacturer’s instructions. RNA concentration was measured with the use of NanoDrop Spectrophotometer (NanoDrop ND-1000; Thermoscientific, Waltham), and RNA quality was determined by TapeStation 4200 (Agilent, Santa Clara).

After the chest cavity was exposed, cardiac puncture was performed through a 25 gauge needle into a sterile 1 ml polypropylene syringe. After the needle was removed, the blood was expelled into Becton-Dickinson K2E Microtainer Tubes, which contained potassium EDTA. Anticoagulated blood was split into a sample that was placed on ice for same-day delivery to the veterinary hematology laboratory and a sample intended for RNA extraction which was transferred to an Invitrogen RiboPure tube with DNA/RNA Later and this suspension was stored at –20 °C. RNA was isolated using the Invitrogen Mouse RiboPure-Blood RNA Isolation Kit. RNA concentration was determined on a NanoDrop microvolume spectrophotometer (ThermoFisher) and quality was assessed on an Agilent Bioanalyzer 2100.

To generate intratibial bone metastasis tumors, mice were s.c. injected with 1 mg/kg of the analgesic buprenexSR by the University of Colorado Anschutz Veterinarian Technicians. The mice were anesthetized under isoflurane for the procedure. Tibias were injected using 27-gauge needles (Easy Touch, 827555) containing 1 × 10⁵ B6 MMTV-PyMT Bone Clone cells in 20 μL PBS or PBS alone as a sham control in the other tibia. To generate mammary fat pad tumors, mice were anesthetized under isoflurane and were then injected with 1 × 10⁵ B6 MMTV-PyMT Bone Clone cells in 100 μL PBS into the fourth mammary fat pad on both sides of the mouse using 27-gauge needles.
Mice were monitored by weekly weight measurement, observation, and palpation. Once tumors were palpable, measurements of tumor length and width were collected every 3–5 days with calipers, and animals were sacrificed after tumors reached the maximum measurement of 2.5 cm in any direction. Tumor volume was calculated by volume = (length × width²)/2. Peripheral blood from the heart was collected in RNAlater after sacrifice for later blood RNA isolation using the Mouse RiboPure Blood RNA Isolation Kit (Invitrogen, AM1951). Tissues including heparin-cleared lungs, spleens, and tibias were collected for histological analysis.