Revertaid m mulv

Total RNA was isolated from randomly chosen subgroups of liver and BAT samples of the transplanted LDL receptor knockout recipients obtained at sacrifice, i.e. after the 20 week WTD challenge. cDNA was synthesized using RevertAid M-MuLV reverse transcriptase according to the manufacturer’s protocol (Thermo Scientific, USA). Relative mRNA expression levels were measured on a 7500 Fast Real-Time PCR system (Applied Biosystems) using SensiMix SYBR green technology. The average expression of the housekeeping genes GAPDH, HPRT and 36B4 was used as a reference for calculation of the relative expression levels of genes of interest. Primer sequences are available upon request.

The PBMCs derived from allergic patients were cultured with or without Der p2 or LPS for 3 days, and the cell pellets were then collected to extract RNA for Cε mRNA expression and supernatant for cytokine analysis. Total RNA from cells was extracted using the RNeasy mini kit (Qiagen, Valencia, CA, USA). The first standard cDNA was synthesized by the RevertAid M-MuLV reverse transcriptase (Thermo Fisher Scientific, Uppsala, Sweden) according to the manufacturer’s protocol. The cDNA then served as a template in a PCR using the G-Storm PCR machine (GS4822, Somerton Biotechnology Centre, Catcombe, UK). The IgE synthesis from B-cells carries out from the IgM to isotype IgG, which mRNA expression from Cμ gene to Cε gene. Detection of excision circular transcripts from direct Cμ gene to Cε switching events was performed using a forward primer in Iε (5'-CGTCTTCCCCTTGACCCGCTGCTG-3') together with a reverse primer in Cμ (5'-CACGTCCATGACCTGCCCGTCCTC-3'). The amplification cycles were as follows: 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s. The PCR products were then subjected to electrophoresis on a 2% agarose gel after 30 cycles and the electrophoresis products were visualized by ethidium bromide staining. The mRNA of GAPDH was used to control sample integrity and loading.

RNA extraction from tissue was performed as recommended by the manufacturer (PEQLAB) using peqGOLD RNAPure. One microgram of RNA was used for reverse transcription (RT)-PCR. We used RevertAid M-MuLV (Moloney murine leukemia virus) RT (200 U/μl) provided with 5× reaction buffer and deoxy (d) ATP, dCTP, dGTP, dTTP (10 mm each), and random hexamers (50–200 ng; Thermo Fisher Scientific). Two microliters of cDNA were used for amplification of the housekeeping gene β-actin; GlyR α1, α2, and β; gephyrin, and GlyT1 (95°C for 5 min; 25 cycles at 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and 72°C for 10 min).

Expression of rol C gene was analyzed by semiquantitative reverse transcriptase PCR. Total RNA was extracted from transformed and untransformed (WT) plants according to the reported procedure (Shirzadegan et al. 1991 (link)). DNAse treatment was given to the extracted RNA to confirm complete elimination of DNA. The purity and quantity of extracted RNA was checked by taking absorbance at 260 nm and 280 nm. Then 1–2 μg of RNA was reverse transcribed at 42 °C in a 20 μl reaction mixture containing 200 units of RevertAid M-MuLV (Moloney murine leukemia virus) reverse transcriptase (Thermo Scientific #K1622), according to the manufacturer’s instructions in the presence of RNAse inhibitor. PCR was carried out with rol C gene primers by using 1–2 μL of cDNA reaction mixture as template. The PCR products were evaluated on 1.5 % agarose gel containing ethidium bromide and photographed under UV. The band on agarose gel indicated the presence of mRNA and intensity of the bands indicated the quantity of mRNA levels.

The presence of NDV in the allantoic fluid was further reconfirmed by RT-PCR. In brief, RNA was extracted from HI-positive allantoic fluids using a Qiagen RNeasy Extraction Kit (Hilden, Germany) following the protocol provided by the manufacturer. The extracted RNA was subjected to two steps RT-PCR using Revert Aid (M-Mulv) (Thermo Fisher). The primer Fusion-Forward (5′-TTGATGGCAGGCCTCTTGC-3′) and Fusion-Reverse (5′-GGAGGATGTTGGCAGCATT-3′) were used to amplify a 363 bp genome fragment containing a part of F gene of NDV [14 (link)]. The RT-PCR cycle program was reverse transcription at 50 °C for 30 min, initial denaturation at 94 °C for 5 min, 35 cycles with denaturation at 94 °C for 30 s, annealing at 56 °C for 1 min, extension at 72 °C for 2 min and final extension at 72 °C for 5 min. The amplified RT-PCR products were analyzed by 1.5% agarose gel electrophoresis containing ethidium bromide (0.5 µg mL⁻¹) and visualized in an image documentation system.

RNA was extracted from cultured B cells by using Trizol reagent according to manufacturer's instructions (Invitrogen) after which cDNA was generated using RevertAid M-MuLV reverse transcriptase according to the instructions of the manufacturer (Thermo Scientific). Quantitative gene expression analysis was performed using Power SYBR Green Master Mix on a 7500 Fast Real-Time PCR system (Applied Biosystems). Gene expression was normalized to housekeeping genes. Primer sequences are available in Supplementary Table 1.