Rotor gene q 5plex hrm

Total RNA was isolated from 1 × 10⁶ cytokine-polarized macrophages using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA), and quantified in a NanoDrop OneC spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA). Then, 1 µg of isolated RNA was used to synthesize cDNA for each sample using the RT2 First Strand Kit (Qiagen, Hilden, NRW, Germany) and following the manufacturer’s instructions. The following primers were used: for STAT3 amplification, 5′-GCTTTTGTCAGCGATGGAGT-3′ (forward), 5′-ATTTGTTGACGGGTCTGAAGTT-3′ (reverse) [126 (link),127 (link)], and for the GAPDH housekeeping control, 5′-CTTCACCACCATGGAGAAGGC-3′ (forward), 5′-GGCATGGACTGTGGTCATGAG-3′ (reverse) [128 (link)]. The PCR reaction mixture was performed in a final volume of 12.5 µL, which contained 2X SYBR^® Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA), 250 nM of each primer, and 25 ng of cDNA. The following thermal cycling was employed, an initial step at 95 °C for 10 min; followed by 50 denaturation cycles at 95 °C for 20 s, annealing at 59 °C for 20 s and extension at 72 °C for 20 s. The amplification was performed in a Rotor-Gene Q 5 PLEX HRM (Qiagen, Hilden, NRW, Germany) and was analyzed in the Rotor-Gene 114 Q Software 2.3 (Qiagen, Hilden, NRW, Germany). The 2^−ΔΔCt method was used to determine relative expression.

Total RNAs were extracted from frozen samples using the RNAprep pure Plant Kit (TIANGEN, DP432). Reverse-transcription reactions were performed using the PrimeScript RT reagent kit (Takara, RR047A). The Rotor-Gene Q 5plex HRM (QIAGEN) was used to detect the gene expression patterns by qRT-PCR. The Unigene15793 (encoding tubulin protein) was used as an internal control, and primers used in this research are listed in Table S4.

Real-time PCR and HRM were performed on a Rotor-Gene Q 5-Plex HRM (RGQ) with Rotor-Gene-Pure Detection software v2.2.3 (Qiagen).
If applicable, singleplex reactions for 12S rRNA and cytb were done by using 5 ng of DNA eluted in water. From samples eluted in TE, 1 µL of a 1∶20 dilution was inserted to maintain the ion concentration of the reaction buffer. The reaction mixes contained 1xHRM PCR Master Mix (Qiagen) and 0.2–0.4 µM of each primer (Biomers, Hilden; primers according to [8] (link), [12] (link); Caprinae: 12S rRNA: forward: CGTAAAGCGTGTTAAAGCATCATACT and CGTAAAACGTGTTAAAGCACTACATC, reverse: TGGGTCTTAGCTATGGTGTATCAG; cytb: forward: TATATTGGCACAAACCTAGTCGAA, reverse: GAGGGCTGTGATGATGAATGGG) Water was added to a final volume of 20 µL. Cycling was 95°C for 5 min, 11 cycles with 95°C for 45 s and 66°C to 64°C (0.2°C/cycle) for 80 s, followed by 24 cycles with 95°C for 45 s and 64°C for 80 s. HRM from 65°C to 90°C with 0.1°C/step and a wait of 2 s after each step was performed after amplification. Fluorescence data were collected on the green channel during cycling and on the HRM channel during the melting step. Non-template controls (NTC) were carried along.

Total RNA (1 μg) was reverse-transcribed with QuantiTect Reverse Transcription Kit (Cat No./ID: 205311, Qiagen) according to the manufacturer's instructions. qPCR reactions were conducted on Rotor Gene Q 5plex HRM (Qiagen) in a 10 μl total reaction volume using SYBR Green JumpStart Taq ReadyMix (S4438, Sigma-Aldrich). The mRNA expression of the genes of interest was calculated with reference to two genes, β-actin and GAPDH. Each reaction was run in duplicate and always included a no-template control. The qRT-PCR reaction was programmed to start with a 2 min denaturation step at 95 °C for polymerase activation followed by 40 cycles of 10 sec denaturation at 95 °C, 30 sec of annealing at 72 °C, and 30 sec extension at 60 °C, during which fluorescence was measured. A melting curve was constructed by raising the temperature from 55 °C to 95 °C in sequential 0.5 °C steps for 6 sec. mRNA was assessed using the 2^−ΔCT method.
Primer sequences (5’-3’) were as follows: BCL-2 Fw: GGG GTC ATG TGT GTG TGG AGA G, RVCAT CCC AGC CTC CGT TAT CC; BCL-XL Fw: GGC CAC TTA CCT GAA TGA CC, Rv: AAG AGT GAG CCC AGC AGA AC; β-actin Fw: AAC TGG AAC GGT GGT CAA GGT GAC, Rv: CAA GGG ACT TCC TGT AAC AAT GC; GAPDH Fw: CCC TTC ATT GAC CTC AAC TAC ATG, Rv: TGG GAT TTC CAT TGA TGA CAA C.

PCR amplification of exon 61 of the USH2A gene was performed with genomic DNA with one set of primers (Table 1). All PCRs were performed in a 15 μL reaction mixture containing 30 ng purified genomic DNA, 200 nM of each of the primers and 1× Type-It HRM PCR Kit (Qiagen, Hilden, Germany). PCR reactions were optimized: 5 minutes at 95 °C, followed by 45 cycles as follows: 10 seconds at 95 °C, 30 seconds at 56 °C, and 10 seconds at 72 °C, with detection of the fluorescence on the Green channel. Melting temperature was raised from 65 °C to 95 °C with a ramp of 0.02 °C per second and the detection of fluorescence on the HRM channel. Rotor-Gene Q 5plex HRM was used to perform the reactions (Qiagen, Hilden, Germany). Analysis of the HRM results was conducted using Rotor-Gene Q Series Software, 2.0.2 (Qiagen, Hilden, Germany).
For Sanger sequencing, the PCR products obtained with the HRM analysis were directly used for a sequencing reaction after being purified with Diffinity Rapid Tips (Sigma Aldrich, Taufkirchen, Germany). The ABI Prism 310 capillary sequencer (Applied Biosystems, Foster City, CA, USA) was used for sequencing. The sequencing reaction was performed using Big-Dye terminator chemistry version 1.1 (Applied Biosystems, Foster City, CA, USA). Electropherograms were analyzed with the Sequencing Analysis 5.2.0 software (Applied Biosystems, Foster City, CA, USA).