Rnalater storage solution

Subgingival plaque samples were collected from the clinically identical sites on quadrants 1 and 2 at the baseline and 1-, 2-, 4-, and 6-week follow-up visits by the same periodontist and then coded by a blinded assistant. In brief, the supragingival biofilm was removed by sterile cotton pellets; each site was dried and isolated from the saliva with sterile cotton rolls; 3 sterile paper points were inserted into the periodontal pocket to collect the subgingival biofilm and were immediately suspended in 0.5 mL of RNAlater storage solution (Sigma-Aldrich, St. Louis, United States) and stored at −20°C. In the next stage, DNA was isolated from the samples using commercial kits for DNA isolation, GeneJET (Fermentas). Quantification of F. nucleatum and P. intermedia was performed by using multicolored real-time polymerase chain reaction, TaqMan amplification, and ABI FAST 7500 sequence detection system. The primers, probes, and amplification conditions are shown in Table 1 [23 (link)]. F .nucleatum ATCC 25586 and P. intermedia ATCC 25611 were used as the standard references.

Mice were culled by overdose with intraperitoneal injection of Pentobarbitone. The right lungs were cut in pieces and then stored in microtubes containing either 1 mL of RNAlater Storage Solution (Merck Sigma) or 0.5 mL of RIPA Buffer (Merck Sigma), for RNA or protein extraction respectively. The left lungs were collected, washed in PBS (Gibco, ThermoFisherScientific), inflated with neutral buffered formalin solution (10%; i.e., 4% (v/v) formaldehyde solution), and then stored in tubes filled with 10% neutral buffered formalin solution. After 24 h, these lungs were transferred to 70% alcohol solution for histological processing. They were then embedded in paraffin and 5.0 µm thick sections were obtained using a microtome (Leica RM2255).

All specimens of Atlantic salmon were collected as either recently deceased commercial net-pen mortalities or as live fish dip-netted directly from the commercial or experimental populations. Netted fish were euthanized by a percussive blow to the head and fresh mortalities (denoted by the industry as ‘silvers’) were selected based on gill pallor indicative of death having occurred less than 12 h prior to collection. In all instances, either blood, heart or a combination of spleen, heart and head kidney were aseptically removed for the purpose of PRV-1 molecular screening. Sample type was variable between sites and years; however, in all instances, samples were collected and processed similarly. Blood (100–1000 µL) was obtained from a caudal puncture using a 1 mL syringe and 22-gauge needle and transferred to a 1.5 mL microtube on ice and frozen at -80 °C within 4–6 h. Internal organ samples (50–100 mg) were aseptically excised and preserved in either 1 mL of 70% ethanol or 1 mL of RNAlater™ storage solution (Sigma Aldrich) and frozen at -80 °C within 4–6 h. For histopathology, samples of heart and skin/skeletal muscle (including the lateral line) were preserved directly in 10% Neutral Buffered Formalin and stored at room temperature.

Unstimulated whole saliva samples were collected from healthy volunteers [n = 15, male:female 5:10; age 28.5 ± 4.8 (21–36)]. The participants rinsed their mouth with water before the collection. Saliva samples were collected 10 min later and centrifuged twice (10,000 × g, 5 min) to remove cellular debris and stored at −80°C until used. Six patients with gingivitis and 15 patients with periodontitis were recruited in a private dental practice. Demografic and clinical data of the patients are listed in Table 1. Subgingival plaque and tissue were sampled from the deepest site per quadrant (in case of the gingivitis patients from the mesio-buccal site of the first molar) by inserting each an endodontic paper-point (ISO 055) for 30 s into the pocket until resistance felt. Paper-points were pooled, transferred into a tube and RNAlater™ storage solution (Sigma-Aldrich) was added. Not earlier than 24 h later, gingival crevicular wash-out (GCF) was obtained as previously described (30 (link)). Immediately after sampling, paper-points were stored at −20°C and GCF samples at −80°C until assayed. The total protein concentration in saliva and GCF was determined by bicinchoninic acid assay (Sigma-Aldrich). The patients study protocol was approved by the Ethical Commission of the University of Leipzig. All subjects gave written informed consent in accordance with the Declaration of Helsinki.

Human adrenocortical carcinoma samples were obtained from the 13 patients qualified for adrenalectomy. Normal adrenal glands (N = 13) were collected during the kidney transplantation procedure. After surgical removal, adrenal fragments of size 0.5 cm³ were immediately immersed in RNA Later Storage Solution (Sigma-Aldrich, Missouri, USA) to stabilize and protect intracellular RNA. Then samples were stored at −70°C until RNA isolation, reverse transcription and qPCR analysis were carried out. The research protocol was approved by the Bioethics Committee of PUMS (decision No. 255/15). Written consent was obtained from adrenalectomized patients as well as from kidney donors.

The pupae were separated by sex, kept in 24-well plates and registered every 30 minutes using a Life Web Camera connected to a computer with Timelapse software (available online) and a screen emitting red light. Insects were frozen in liquid nitrogen at the following time points after adult eclosion: 0 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h and 48 h. It was analyzed from 7 to 10 individuals for each strain at each time point.
Before RNA isolation, the fat body (FB) was separated from each individual and treated with an RNAlater Storage Solution (Sigma-Aldrich, Germany). Tissues from each individual were homogenized in 250 µL of QIAzol Lysis Reagent (Qiagen, Germany) using FastPrep® Systems (MP Biomedicals, USA).
According to qPCR publications, seven commonly used candidate reference genes, in this case housekeeping genes, were selected to validate their expression stability (Table 1): GAPDH, G3PDH, EF2, RpL7A, RpL10, TUBα and ACT. Primers were tested by melt curves and product size on a gel analysis. The fat body was chosen for these studies because it is a dynamic tissue that is involved in multiple metabolic functions that are fundamental in the life of holometabolous insects such as Lepidoptera^{24 (link)}. In context of the study on stability of housekeeping gene expression, they take on a special meaning.

Rodent spleens were placed in RNAlater^® storage solution (Sigma-Aldrich, Saint Louis, MO, USA) then stored at −20 °C until further analysis. Genomic DNA was then extracted from the spleen using the DNeasy® 96 Tissue Kit (Qiagen, Germany). Spleen DNA samples were screened for the presence of bacteria using universal primers (16S-V4F [GTGCCAGCMGCCGCGGTAA] and 16S-V4R [GGACTACHVGGGTWTCTAATCC]) targeting the hypervariable region V4 of the 16S rRNA gene (251 bp) via Illumina MiSeq (Illumina) sequencing. The V4 region has been proven to have excellent taxonomic resolution at the genus level [37 (link)]. A multiplexing strategy enabled the identification of bacterial genera in each individual sample. We followed the method described in Kozich et al. [38 (link)] to perform PCR amplification, indexing, pooling, de-multiplexing and finally taxonomic identification using the SILVA SSU Ref NR 119 database as a reference [39 ] (http://www.arb-silva.de/projects/ssu-ref-nr/). We then used the trimming strategy of Galan et al. [39 ,40 (link)] in order to clean the raw data set and to estimate reliable rodent positivity for bacteria.