Rnalater solution

Fresh core needle tumor samples (pancreas cancer, n = 1; cancer of the vulva, n = 1; colon carcinoma, n = 2, and BC, n = 88) and FFPE tumor samples (BC, n = 7) were obtained using a 16 G × 100 mm or 18 G × 100 mm core needle biopsy instrument from patients who were treated at Universitair Ziekenhuis Brussel (UZ Brussel) from December 2017 to January 2020 and who gave informed consent. Fresh CNB were collected in 50 mL tubes (Sarstedt, 62.547.254, Berchem, Belgium) containing 5 mL RNAlater^TM solution (Sigma-Aldrich, R0901, Overijse, Belgium,). Samples were stored at 4 °C for a maximum of 1 month before further processing. The project followed the Helsinki Declaration and was approved by the ethics council of the UZ Brussel (2017/344 and 2017/400).

Ad libitum feeding was maintained until just before the dissection in all piglets. At the dissection, pigs were intraperitoneally anesthetized with sodium pentobarbital (Somnopentyl; Kyoritsu, Tokyo, Japan). All dissections started at 11:00 a.m. and collection of blood samples from all location of a piglet were finished within 5 min after confirmation of deep anesthesia. Briefly, the abdominal wall of pigs was incised along midline, blood was quickly collected from the cecal, portal, and abdominal veins, and the animals were euthanized by exsanguination. Afterward, the entire intestine was removed, the large intestine separated, and the cecal digesta collected. The cecum was washed several times with sterilized saline, and its middle section of mucosa soaked in RNA-later^® solution (Sigma, Tokyo, Japan), and stored first at 4 °C for 24 h, then at −80 °C until use. Blood samples were centrifuged at 1750× g for 10 min at 4 °C and serum was collected. Serum and digesta samples were stored at −80 °C until use.

All animal procedures were approved by the Institutional Animal Care and Use Committee of the VA Boston Healthcare System. Male C57/BL6 (RRID:IMSR_JAX:000664) mice were obtained from Jackson Labs (Bar Harbor, ME, United States). Between 8 and 14 weeks of age, animals were euthanized by CO₂ asphyxiation, and organs were quickly dissected and either stabilized in RNA-Later solution (R0901, Sigma-Aldrich, St Louis, MO, United States), or embedded in OCT compound, immediately snap frozen and stored at −80°C.

The Ornithodoros moubata ticks were obtained from a pathogen-free laboratory colony, maintained in the IRNASA-CSIC (Salamanca, Spain), which was established in the 1990s from specimens kindly donated by Dr Philip Wilkinson (Institute for Animal Health, Pirbright, UK). Ticks were kept at 28°C, 85% relative humidity, 12 h light/12 h dark photoperiod and regularly fed on New Zealand White rabbits. For tick feeding, rabbits were immobilised with their abdomen facing up and their skin shaved. Ticks were allowed to feed by placing them inside a plastic cylinder fixed to the rabbit skin with surgical tape. Ticks feed during approximately 60 minutes, and after detaching themselves they were collected. The rabbits were not treated with any anaesthetic or tranquilizer drug in order not to interfere with the physiology of the ticks.
Salivary glands (SG) were obtained from newly moulted 3-month-old female ticks in three different physiological states: unfed (SG0) and at 7 and 14 days after feeding (SG7 and SG14, respectively). Tick dissection and salivary gland extraction were performed in cold (4°C) phosphate-buffered saline (PBS) pH 7.4 treated with 0.1% diethyl pyrocarbonate (DEPC), and the SG were immediately stabilised in RNAlater solution (Sigma). For each physiological state, two replicate samples of 20 pairs of SG per sample were collected and used for RNA isolation.

This study was performed on 28 patients who were referred to Taleghani Hospital, Tehran, Iran, for treatment and/or diagnosis. After a colonoscopy and confirmation of the results by a pathologist, these patients were further referred by a gastroenterologist for genetic testing in this research project. Patients were asked to answer demographic questions on a questionnaire. Thereafter, specimens were obtained individually from the patients from their seemingly healthy margins, during a colonoscopy by a gastroenterologist specializing in CRC, and placed in RNAlater solution (Sigma, Germany). The samples were immediately transferred to liquid nitrogen and stored until the RNA extraction. Exclusion criteria included patients with T1 cancer treated by endoscopic polypectomy, patients who received neoadjuvant chemotherapy, and patients with synchronous or metachronous invasive cancers originating from the colorectum or other sites [21 (link)].

Myometrium was collected from the inferior margin of the incision site of women delivering by caesarean section, at the Royal Infirmary, Edinburgh. Written and informed consent was obtained according to the ethical approval and governance granted to the Edinburgh Reproductive Tissues BioBank by the West of Scotland Research Ethics Committee 4 (REC reference: 09/S0704/3) until 30/09/2014; and consequently by the East of Scotland Research Ethics Service Tayside Committee on Medical Research Ethics B (REC reference:13/ES/0126). Upon collection, samples were placed into RNAlater solution (R0901 Sigma-Aldrich) for 24 hours at 4 °C, then taken out and stored at −80 °C prior to use. Inclusion criteria was singleton pregnancies that were term (>37 weeks of gestation) or preterm (<37 weeks of gestation) whilst exclusion criteria were age under 16 and any blood borne infections. Women either underwent an elective pre-labour caesarean section (no labour group) or an emergency CS in labour due to maternal and/or fetal indications (e.g. delay in labour, pre-eclampsia, fetal distress). Labour was defined as regular uterine contractions with cervical dilation.

The pregnant females from the respective LP and HP groups were deeply anesthetized with diethyl ether and the embryos/foetuses of varied embryonic ages (E11, 14, 16 and 18) were excised surgically with atraumatic measures. The brain tissues of the embryos were micro dissected. Half of the tissues were stored in RNA Later solution (Sigma, USA) for RNA isolation, while other half was processed for cryosectioning. Embryonic tissues for cryosectioning were fixed in 2% paraformaldehyde (PFA) in 0.01 M PBS, pH 7.4 for 24 h, followed by 3 times washing in phosphate buffer and subsequently cryoprotected with sucrose gradients (10%, 20% and 30%).