Proteinase k

Saliva samples were processed at the biosafety security level 3 laboratory facilities at FISABIO-Public Health as follows: After thawing the samples at room temperature, a 200 uL aliquot was taken for RNA extraction, and the remainder was immediately stored at −80°C for the assay in cell lines. RNA extraction was carried out with the fully automated eMAG platform (bioMérieux, France) following the manufacturer's instructions for saliva samples, preceded by lysis with proteinase K (Epicentre) during 20 minutes at 56 °C. Then, the a multiplex RT-qPCR test was performed to detect the SARS-CoV-2 E-gene and the human RNAse-P gene as a sample and extraction control based in the WHO-Charité and U.S. CDC assays [31 (link),33 ] following the protocol details described by Ferrer et al. [19 (link)]. Two replicates per sample of the extracted RNA were performed and virus copies were normalized by mL of saliva.

Frozen tissues were pulverized to form a powder, and tissue lysates were prepared in Tissue and Cell Lysis Buffer (Epicentre) supplemented with proteinase K (0.5 mg/ml; Epicentre). The mixture was agitated at 1400 rpm for 2 hours at 65°C and centrifuged at 16,000g to remove any debris. The mRNA levels in the supernatant (lysate) were quantified using the QuantiGene 2.0 luminescent-based branched DNA Assay Kit and QuantiGene 2.0 probes against Tie2 and glyceraldehyde-3-phosphate dehydrogenase (Gapdh; Affymetrix) according to the manufacturer’s protocol. Luminescent signal was measured with a Tecan Infinite 200 PRO plate reader. To avoid signal saturation and to ensure that all luminescent signals remained within their linear regions, a standard curve for each tissue and target gene was constructed using samples from PBS-treated mice to determine the optimal dilutions for assay samples. The relative silencing in treated groups was determined by calculating the ratio of target gene luminescence to Gapdh housekeeper gene luminescence. All values were normalized to the target/housekeeper gene ratio from PBS-treated mice. To graph the data, the mean values of the three 7C1-Luc siRNA treatments were used to normalize each of the four 7C1-Tie2 siRNA treatment values. The mean normalized 7C1-Tie2 siRNA treatment values were then plotted along with their SD.

Cells fixed in frozen methanol were washed twice in ddw and RNA purification was performed as previously described (53 (link)) with minor modifications. Briefly, RNA was released from the cells by digestion with Proteinase K (Epicenter) in the presence of 1% SDS at 70°C. Cell debris and proteins were precipitated by centrifugation in the presence of potassium acetate. RNA was then purified from the supernatant using nucleic acid binding plates (96-well, 800 μl UNIFILTER Microplate, GE Healthcare) in the presence of 0.1 mM DTT, eluted in 1 mM DTT, and stored at −80°C.

To test the stability of released sRNAs in vitro, a total of 40 μL of concentrated ES from gAF worms was mixed with RNace-IT Ribonuclease cocktail (Agilent) in the absence or presence of 5 μg/μL Proteinase K (Epicentre) and 0.05% Triton X-100 (Merck). Incubations were conducted at 37°C for 30 min. RNA was then extracted with 5X volumes of Qiazol and purified by miRNeasy plasma/serum kit following the manufacturer’s recommendations and eluted in 16 μL of RNase-free water. 10 μL of total RNA was used for analysis by qRT-PCR using miScript II RT System (Qiagen) as indicated above.

Genomic DNA was extracted from fresh (blood) and/or frozen (cirrhotic and tumoral liver) using standard procedures. Briefly, PBS-washed samples, were centrifuged and lysed using “Tissue and cell lysis solution” buffer for the MasterPureTM kit, complemented by proteinase K (5 μl/100 μl buffer) (Epicenter), shaking overnight at 56°C. DNA was extracted using phenol/chloroform/isoamyl alcohol (in proportions of 25:24:1, respectively) in a fast Lock Gel Light Eppendorf tube (Eppendorf), then washed and precipitated. Genomic DNA was quantified using a Qubit ds DNA BR assay kit and a Qubit 2.0 fluorimeter (Invitrogen).