Rna lysis buffer

The quantification of the relative expression of fibrotic markers by qPCR was performed as previously described [21 (link)]. Briefly, LX-2 cells or activated LX-2 cells were cultured in the presence or absence of exosomes for 48 h. The cells were then lysed in 0.2 mL of RNA lysis buffer (Promega, Durham, NC, USA). Total RNA from these lysates was purified using SV 96 Total RNA Isolation System (Promega, Madisin, WI, USA). RNA concentration and purity were measured using TECAN Spark Nano plate (TECAN, Morrisville, NC, USA). cDNA preparation and qPCR were performed as described [76 (link)]. The primers used for qPCR were described in Materials. Each sample was run in duplicate. After the run was completed, a second derivative analysis was performed using the raw data to determine the mean Cp (Crossing point-PCR-cycle) for each sample. For each gene expression, expression of beta-actin served as an internal control. Relative mRNA expression was determined by Pfaffl analysis (EΔCp target/EΔCp reference), in which primer efficiency E = 10^(−1/slope) and ΔCp = mean Cp of sample − mean Cp of the cells treated with PBS (Control).

Total RNA was extracted from 50–100 mg of grounded frozen tissue specimens by the miRNeasy Mini Kit (QIAGEN, Hilden, Germany) in compliance with the supplier’s instructions. Each sample was put in 600 μL RNA lysis buffer (Promega, Madison, Wisconsin) until complete digestion of the tissues occurred. The final extracted RNA volume was 40 µL.

At the end of hMSC culturing on GelMPs or in GelMA, 0.2 mL/well of RNA lysis buffer (Promega, Madison, WI, USA) was added on cells and frozen down at −80 °C. Then, the samples were thawed, and DNA was quantified using the Helixyte Green dsDNA Assay Kit (AAT Bioquest, Pleasanton, CA, USA).

RNA from biofilms was isolated using the SV Total RNA Isolation System kit (Promega). RNA was extracted from strains grown O/N at 37°C in LB and then spotted on LB-NaCl agar plates and incubated for 48 h at 30°C. Cells from each spot were then resuspended in 100 μL TE containing 50 mg/mL lysozyme and were homogenized by vortexing. Seventy-five microliter of RNA Lysis Buffer (Promega kit), followed by 350 μL RNA Dilution Buffer (Promega kit) were added to the cell suspensions, which were then mixed by inversion. Samples were incubated at 70°C for 3 min and centrifuged at 13,000 g for 10 min. The supernatant was mixed with 200 μL 95% ethanol and was then loaded on to the spin columns provided by the kit. The columns were washed with 600 μL RNA Wash Solution. DNase mix was prepared following the Promega kit protocol and 50 μL were directly added on the column membrane. After a 30 min incubation, 200 μL DNase Stop Solution was added and samples were centrifuged for 30 s. Columns were washed with 600 μL RNA Wash Solution followed by 250 μL RNA Wash Solution, and then centrifuged again for 1 min to dry. RNA was eluted using 100 μL of nuclease-free water. RNA quantification was performed using the Qubit RNA High Sensitivity assay kit (Q32852).

Tenocytes (2 × 10⁴/well) were added to each well of 48-well plates containing scaffolds. There were two sets of samples. After culturing for 24 h, the medium was removed from all samples. Fresh culture medium was added to each well and incubation continued. After 24 h, the medium was removed from each well of one set of samples. Cells in the wells were lysed with 0.2 mL/well RNA Lysis buffer (Promega, Durham, NC). The RNA lysates were stored at −80 °C. The cells in the second set of samples continued to culture, and the medium was changed every three days. On Day 7, the medium was removed from each well of the second set of samples. Cells in the wells were lysed with 0.2 mL/well RNA Lysis buffer.

Cells were lysed using RNA lysis buffer from Promega, Leiden, the Netherlands. Total RNA was robotically isolated using Maxwell® 16 simply RNA tissue kit (Promega) and quantified using a Nanodrop ND-1000 UV-visible spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). RNA was reverse-transcribed and cDNA was amplified by qPCR (Bio-Rad, Veenendaal, the Netherlands). Relative gene expression compared to reference genes Ribosomal Protein L13a (RPL13A) and ATP synthase, H⁺ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B) were calculated according to the standard curve method. Reference genes were selected out of 8 candidate reference genes using the “Genorm” software (Genorm; Primer Design, Southampton, UK). Primer pairs are presented in Table 1.