Rnalater stabilizing solution

Tumor and paired nontumor liver tissues were collected from 24 HCC patients at the National Cancer Institute (INT) of Naples “G. Pascale”. Samples were stored in 0.5 mL of RNAlater stabilizing solution (Ambion, Austin, TX, USA) for 16 h at 4 °C and then transferred and preserved at −80 °C. Stored samples were used to perform an RNA-seq analysis.
The transcript levels of overexpressed proteins were also evaluated on the Gene Expression database of Normal and Tumor Tissues 2 (GENT2) database (http://gent2.appex.kr (accessed on 18 March 2021)), a search platform for gene expression patterns across different normal and tumor tissues compiled from public gene expression datasets.

Animals (n = 8 per time point) were randomly assigned to each group. At t = 0, 45 min, and 24 h post-surgery, mice were anesthetized and euthanized, hearts were harvested and the left ventricle was dissected to maximally provide the myocardium known to be at risk in ischemic hearts. Myocardium samples were placed in RNAlater stabilizing solution (Ambion, Thermo Fisher Scientific) and stored at −80°C until use.
RNA was extracted by Tripure reagent solution (Roche), treated with Proteinase K (Qiagen) and purified by RNeasy Mini kit (Qiagen) where DNase I (Qiagen) is treated on column. RNA purity, quantity and integrity were assessed both by spectrophotometry (NanoDrop ND-1000, NanoDrop Technologies) and nanoelectrophoresis (2100 Bioanalyzer, Agilent Technologies). RNA purity: A_260/280 ∼ 1,8 and A_260/230 ∼ 2 and RNA integrity number: 8–10.

After sacrificing and BAL collection, the lungs were removed, cut and transferred to RNALater stabilizing solution (Ambion, Life Technologies, CA, USA), and stored at −80 °C. Total RNA (totRNA) from the RNAlater‐stabilized lung samples was isolated and purified by phenol/chloroform isolation method according to the instructions by Bioline Reagents. In brief, tissue samples in RNALater were thawed and moved to the lysing matrix tubes with ceramic spheres (D, 1.4 mm) (MP Biomedicals, Illkirch, France), containing 1 mL of TRIsure reagent (Bioline Reagents, Ltd., London, UK). Samples were homogenized in a FastPrep FP120 homogenizer (BIO 101, Thermo savant, Waltham, MA, USA). RNA was separated with chloroform, precipitated with isopropyl alcohol, washed with 75% ethanol, re‐dissolved into DEPC‐treated water, and stored in −80 °C. Quantity and quality of the mRNA were measured with NanoDrop spectrophotometer (ND‐1000, Thermo Fisher Scientific Inc., Wilmington, NC, USA) and Bioanalyzer (Agilent Technologies, USA). Two to three samples from the same experimental group were pooled, and independent pools of samples with RIN >8.3 were used for DNA microarray analysis.

Participants received a fecal collection kit containing a stool collection container, two Sarstedt feces tubes (Numbrect, Germany) filled with 2.5 mL of RNAlater stabilizing solution (Ambion, Inc., Austin, Texas), two FOBT cards (Hemoccult II Elite Dispensapak Plus, Beckman Coulter, Brea, California), and a wooden applicator stick. After the feces were collected in the container, the participants were instructed to add a scoop of the feces into each of the two Sarstedt feces tubes and spread a small amount of feces onto the two FOBT cards. Approximately half of the participants provided a fecal sample at the clinic and half collected their samples at home. For participants collecting fecal samples in the clinic, one Sarstedt tube and one FOBT card were immediately returned to the laboratory and frozen at − 80 °C (day-0). For those collected at home, samples were delivered to the laboratory within a few hours after collection and then one Sarstedt tube and one FOBT card were frozen at − 80 °C (day-0). Whether collected on site or at home, each participants’ second Sarstedt tube and FOBT card were left room temperature for 96 h (day-4) before being frozen at -80 °C.

Across the entire Mojave Desert, 20 populations of wild tortoises (n = 419) were sampled during their active seasons (April–June) in 2010–2012 (described in Sandmeier et al., 2018 ; Weitzman et al., 2017 ). Individual tortoises were evaluated for presence (0 or 1) and severity (scored 0–6) of signs of disease (Sandmeier, Weitzman, et al., 2017 (link); Weitzman et al., 2017 ). Nasal lavage samples were collected from each tortoise by rinsing 3 mL of sterile saline solution through the nares. The lavage samples were preserved in RNAlater stabilizing solution (Ambion Inc.) and placed on ice until they could be frozen within 12 h of collection (Weitzman et al., 2017 ). DNA was then extracted from 500 μL of the samples with the Qiagen DNeasy Blood and Tissue Kit (Qiagen Inc.) and stored at −20°C (Weitzman et al., 2017 ). One‐hundred forty samples from 17 local populations tested positive for M. agassizii by qPCR (Sandmeier, Weitzman, et al., 2017 (link); Weitzman et al., 2017 ) and had enough volume of DNA sample remaining to be tested by four separate additional assays: three assays for each of the putative sialidase genes, and one assay for the quantification of M. agassizii (Braun et al., 2014 (link)).

This study was performed in pretreatment samples of CHB patients who participated in two independent investigator-initiated studies, which were described in detail previously (Takkenberg et al., 2013 (link); de Niet et al., 2017 (link)). From the first cohort, consisting of HBeAg-negative CHB patients with HBV-DNA levels below 20,000 IU/mL, 150 plasma and 97 liver samples were available for analysis. From the second cohort, consisting of HBeAg-positive or -negative patients with HBV DNA levels >17,182 IU/mL, a total of 32 liver samples were available for analysis. Control liver tissue was obtained from 13 HBV negative patients undergoing surgical liver resection and from the resected liver tissue of the non-affected, tumor free margin surrounding the pathology was stored in RNAlater stabilizing solution (Thermo Fisher Scientific, Waltham, MA, United States) (Stelma et al., 2017 (link)). Control plasma samples were obtained from 10 healthy volunteers. Baseline characteristics of all cohorts are shown in Table 1.

This study was performed in pretreatment samples of CHB patients who participated in a prospective randomized controlled intervention study, which was previously described in detail [17 (link)]. This cohort consisted of 151 HBeAg-negative CHB patients with HBV-DNA levels below 20,000 IU/mL, of which 97 matching plasma and liver biopsy samples were available for analysis. Control plasma samples were obtained from 10 healthy volunteers. Control liver tissue from 13 HBV negative patients undergoing surgical liver resection was obtained from the resected liver tissue of the non-affected, tumor free margin surrounding the pathology [18 (link)]. Upon further use all liver samples were stored in RNAlater stabilizing solution (Thermo Fisher Scientific, Waltham, MA, USA). Baseline characteristics of the cohorts are shown in Table 1.