Dnase 1 amp grade

Placentas obtained from pregnant women with PE and NT were submitted to analysis of the protein-encoding genes LC3-II, beclin-1, and mTOR. Total RNA was extracted from the placentas with the Total RNA Purification Kit (NorgenBiotek Corp., Canada) according to the manufacturer's protocol. After extraction, 1 µg of total RNA was incubated with DNase I Amp Grade (Invitrogen, ThermoFisher Scientific, USA), and measured by Qubit^® Fluorometric Quantitation (ThermoFisher Scientific). Subsequently, complementary DNA (cDNA) was synthesized using the ImProm-II^TM Reverse Transcription System (Promega, USA) according to the manufacturer's protocol. Quantitative real-time PCR (qPCR) was performed using RT GoTaq^® qPCR Master Mix (Promega) according to Matias et al. (20 (link)). A 7500 Fast Real-Time PCR System (Applied Biosystems - ThermoFisher Scientific, USA) was used for the analysis. For normalization, GAPDH was used. The primers used in the experiments are listed in Table 1. The differential expression calculation of selected genes was carried out by the data processing method compared with a standard curve (21 (link)). To analyze relative gene expression, RNA expression levels in all samples were standardized on a single RNA sample, which was set to a value of 100.

Floral buds were collected from stage 1, whereas the callus and gynostemium (including mentum and stelidia, but excluding the anther cap with pollinia) were dissected and collected from stage 5 (Fig. 3). A maximum of 100 mg of each sample was transferred into sterile 2.2-mL micro-centrifuge tubes, together with a 7-mm glass bead (Assistent), and stored at − 80 °C. The frozen samples were grinded by using a TissueLyser II (QIAGEN). RNA was extracted by using a RNeasy Plant Mini Kit (QIAGEN), under RNase-free conditions. DNase I Amp Grade (Invitrogen 1U/μl) was applied to the extracted RNA to digest single- and double-stranded DNA. To assess the quantity and quality of the samples for cDNA synthesis, they were measured with a nanodrop (NanoDrop 2000c, ThermoFisher), whereas for sequencing, they were measured by Agilent 2100 Bioanalyzer Systems (Agilent Technologies). The quantity of the RNA samples analyzed was at least 50 ng/μL RNA in 50 μL volume, the quality as assessed by the RNA integrity Number (RIN) was at least 7. All RNA samples with a RIN < 7 were discarded. Samples used for further downstream experiments were stored at − 80 °C.

All 19 M. tuberculosis strains were subcultured in 7H9 medium with ADC supplement, and collected for RNA extraction. For the nine MDR isolates, RIF and INH were added to these cultures individually at subinhibitory concentrations (half of the MIC), incubated at 37°C for 25 days, and collected for RNA extraction. Total bacterial RNA was isolated using Trizol reagent according to the manufacturer’s instructions. The quality and integrity of the total RNA was assessed using a nanophotometer (Implen, Munden, Germany) and agarose gel electrophoresis. After treatment with DNase I (amp grade; Invitrogen), the lack of DNA contamination of the RNA samples was confirmed by polymerase chain reaction (PCR) amplification of rpoB directly from RNA. The forward and reverse primers are listed in S1 Table. A 50-μL aliquot of the PCR mixture (Kangwei Biotechnology, Beijing, China) was used, and the PCRs were denatured at 94°C for 5 min and subjected to 35 cycles at 94°C for 30 s, 62°C for 30 s, and 72°C for 30 s, followed by a final extension at 72°C for 10 min. RNA (1.5 μg) was reverse transcribed according to the manufacturer’s recommendations (Transgen Bio-Technology Company, Beijing, China), and the thermal cycling conditions were as follows: 25°C for 10 min, 42°C for 60 min, and 85°C for 5 min. The cDNA was maintained at −20°C. Two cDNA preparations were made for each strain.