Cfx96 rt pcr detection system

The kidney tissue was homogenized using TRIzol (Ambion, Austin, TX, USA). The total RNA was separated using TRIzol/chloroform, precipitated with isopropanol, and washed using ethyl alcohol in order. The NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine RNA quantity and quality. The concentration and purification of RNA were estimated by measuring the absorbance at 260/280 and 260/230 nm using spectrophotometry. Purified RNA of each sample was used for cDNA synthesis using a Prime Script™ RT reagent kit (Takara, Shiga, Japan). PCR amplification was performed with SYBR green to detect relative mRNA expression using the CFX96TM RT-PCR detection system (Bio-Rad, Hercules, CA, USA). The primer sequences for PCR were as follows; renin (NM_012642.4) forward primer, 5′-TGCTAAAGGAGGAAGTGTTT-3′; renin reverse primer, 5′-TGATGCTCACGTAGTGAAAG-3′; GAPDH (NM_017008.4) forward primer, 5′-GTCGGTGTGAACGGATTTG-3′, GAPDH reverse primer, 5′-TCCCATTCTCAGCCTTGAC-3′. Each value was normalized to GAPDH, and the relative expression levels of the genes were calculated using the delta-delta threshold cycle (ΔΔCt) method compared to the SHR.

Primer pairs (Table 2) were designed using the coding sequences (CDS) of the carvone and limonene biosynthesis genes of M. longifolia. The qRT-PCR experiments were carried out using the SYBR green master mix and the CFX96TM RT-PCR Detection System (Bio-Rad in Hercules, California, USA). The PCR reaction mixtures (10 μl) contained SYBR Green Master Mix 5.0 μl, Primer 1.0 µM (Saha Gene, Hyderabad, Telangana, India), appropriately diluted cDNA as a template, and the final volume of 10 μl was made up of MQ water. The thermoprofiles of the qRT-PCR reactions were included by preincubating at 95°C for 10 min, followed by 45 cycles of 3-step amplification (95°C for 10 s, 55°C for 10 s, and 72°C for 15 s) and melting. The reaction was normalized by using the primers for the 5.8 s gene as a reference. The fold change was then calculated using the 2^−ΔΔCT approach utilizing the threshold cycle (CT) (Ahmed et al., 2022 (link)).

Total mRNA was isolated from the kidney with TRIzol (Ambion, Austin, TX, USA) and PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The concentrations of RNA were determined using a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). The purified RNA of each sample was subjected to cDNA synthesis using Prime Script™ RT reagent kit (Takara, Shiga, Japan). PCR was performed with SYBR green to measure the renin mRNA expression using the CFX96TM RT-PCR detection system (Bio-Rad, Hercules, CA, USA). The sequences of primer were as follows: renin (NM_012642.4) forward primer, 5′-TGCTAAAGGAGGAAGTGTTT-3′; renin reverse primer, 5′-TGATGCTCACGTAGTGAAAG-3′; GAPDH (NM_017008.4) forward primer, 5′-GTCGGTGTGAACGGATTTG-3′; GAPDH reverse primer, 5′-TCCCATTCTCAGCCTTGAC-3′. The relative gene expression of renin was quantified using GAPDH. The data were analyzed using the Bio-Rad CFX-Manager software 3.1 (Bio-Rad, Hercules, CA, USA).

CD33⁺ myeloid cells were purified from PBMCs as described above. Total cellular RNA from CD33⁺ cells (pooled from 6 chronic HCV patients and 6 HS) was isolated using the miRNeasy Mini kit (Qiagen, Valencia, CA, USA). The RNA quality and quantity were analyzed using a BioPhotometer spectrophotometer UV/VIS, and RNA integrity was determined using gel electrophoresis. LncRNAs were analyzed using the Arraystar gene array service (Rockville, MD. The miScript miRNA array was performed by Qiagen [17 (link)]. To validate the results for up- or downregulated miRNAs by real-time RT-PCR, cDNA was generated from total RNA by the Taqman advanced miRNA cDNA synthesis kit and the High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific, Logan, UT, USA). The miRNA expression levels were assessed by RT-qPCR using Taqman^® fast advanced master mix (Thermo Scientific) and the CFX96TM RT-PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The miRNA levels were determined using the 2^−ΔΔct relative quantification method and were normalized to U6 RNA (SNORD61) level as an internal control.

We analyzed gene expression patterns of Zt80707, Zt89160, Zt103264 and Zt110804 in Z. tritici using a qRT-PCR experiment. Total RNA was extracted from fungal axenic cultures (grown for 72 h in YMS medium at 18°C and 140 rpm) and from freeze-dried leaf tissue infected with Z. tritici (4, 7, 14 and 28 dpi) using the TRIZOL reagent (Invitrogen), following the manufacturer’s instructions. Three biological replicates were sampled from axenically grown cultures and from each time point of infection. The samples were crushed in liquid nitrogen and 100 mg was used for RNA extraction and cDNA synthesis. The cDNA samples were used in a qRT-PCR experiment employing the iQ SYBR Green Supermix Kit (Bio-Rad, Munich, Germany), GSPs (S1 Table) and an annealing temperature of 59°C. PCR was conducted in a CFX96 RT-PCR Detection System (Bio-Rad) with the constitutively expressed control gene Zt99044, a Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [33 (link)]. A Mann-Whitney U test was applied to test the significance of different gene expression levels.