Rotor gene q pcr cycler

To confirm GS gene deletion, total RNA was isolated from 1 × 10⁶ viable cells using Direct-zol RNA Kit (Zymo Research, USA) with DNase treatment, following manufacturer’s instructions. RNA (800 ng) was converted to cDNA using a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, USA). For Quantitative PCRs (qPCRs), one microliter of the 4 × diluted cDNA was used as a template in 10 µL reaction using the SensiFAST SYBR Hi-ROX Kit (Bioline, UK). Samples were measured in quadruplicates on a Rotor-Gene qPCR cycler (Qiagen, Germany) as previously described^{37 (link)}. The primer pairs specific for CHO, i.e., GAPDH (glyceraldeyde-3-phoshate dehydrogenase), Alpha-(1,6)-fucosyltransferase (FUT8) and three GS genes are listed in Supplementary Table S2. Relative quantification of gene expression level was normalized to GAPDH (for mRNA) or FUT8 (for gDNA) for calculation of fold change (FC) by $2^{-\mathrm{\Delta }\mathrm{\Delta }{\text{C}}_{\text{T}}}$ method^{38 (link)}.

RNA was extracted from the non-differentiated iPS cells, primary epithelial cells, hAD-MSC-derived myofibroblasts, and primary endothelial cells with the Zymo Research Quick-RNA Miniprep Kit following the manufacturer’s instructions. The RNA was converted to cDNA with the Qiagen QuantiTect Reverse Transcription Kit. Finally, reactions were set up in triplicate with the Qiagen QuantiTect SYBR green PCR kit. The RT-qPCR reactions were performed in a Rotor-Gene Q PCR Cycler (Qiagen, Hilden, Germany). Analysis of the relative fold gene expression of each sample was performed by calculating the ∆∆CT for each primer set with cDNA from the undifferentiated iPS cells as the control.

IMX2538 (35-deletions prototrophic strain) was sequenced using 300-bp paired-end sequencing reads prepared with the MiSeq reagent kit v3 on an Illumina MiSeq sequencer (Illumina, San Diego, CA). To this end, extracted DNA was mechanically sheared to 550 bp with the M220 ultrasonicator (Covaris, Wolburn, MA) and subsequently the TruSeq DNA PCR-Free Library preparation kit (Illumina) was employed to make a six-strain library. The samples were quantified by quantitative PCR on a Rotor-Gene Q PCR cycler (Qiagen) using the KAPA library quantification kit (Kapa Biosystems, Wilmington, MA). Library integrity and fragment size were determined with a Tapestation 2200 system (Agilent Technologies). Sequencing reads were mapped onto the CEN.PK113-7D (163 (link)) reference genome using the Burrows-Wheeler alignment (BWA) tool (version 0.7.15) (164 (link)) and further processed using SAMtools (version 1.3.1) (165 (link)) and Pilon (with -vcf setting; version 1.18) (166 (link)) to identify SNPs. The sequence was analyzed by visualizing the generated .bam files in the Integrative Genomics Viewer (IGV) software (version 2.4.0) (167 (link)). Chromosomal copy number was estimated by the Magnolya algorithm (version 0.15) (168 (link)).

Total RNA extraction and purification were carried out using an RNeasy Plant Mini kit (Qiagen, Valencia, CA, United States). To quantitate the mRNA expression levels of genes encoding transporters, heavy metal chelators, and antioxidative enzymes, the quantitative reverse transcription polymerase chain reaction (qRT-PCR) was conducted using a Rotor-Gene Q PCR cycler (Qiagen) and QuantiTect SYBR Green RT-PCR kit (Qiagen) as described previously (Kim and Kang, 2018 (link)). Actin was used as the internal control. The primers used for the qRT-PCR analysis are listed in Supplementary Tables 1 and 2. At least three independent replicates were performed for all experiments with different RNA samples.

The total RNA was isolated from the liver, visceral, and subcutaneous adipose tissue using TRIzol (MRC, Cincinnati, OH, USA) and reverse-transcribed into complementary DNA (cDNA) by TOPscript™ RT DryMIX (Enzynomics, South Korea). A quantitative real-time polymerase chain reaction (PCR) was performed using the TOPreal SYBR Green PCR Kit (Enzynomics). The mRNA levels were detected by real-time PCR using the Rotor-Gene Q PCR cycler (QIAGEN, Hilden, Germany). The level of mRNA was normalized to 18S ribosomal RNA.

qPCR was used to validate the aCGH data. Primer amplification efficiency for each gene was determined. Specific sequences of the reference and target genes were cloned into pGEM-T easy vector (Invitrogen). Known amounts of genomic DNA (from clones D11 and CLB and G strain) and recombinant plasmids containing the sequences of interest were incubated with 10 µL SYBR Green-Based Detection (Applied Biosystems), sense and antisense primers and water to a total reaction volume of 20 µL. The reaction mixture was distributed into 0.2 mL tubes and subjected to 40 cycles of amplification in the Rotor-Gene^® Q PCR cycler (Qiagen) according to the manufacturer’s instructions. The qPCR program was set as follows: initial denaturation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 15 s, annealing and extension at 60°C for 60 s. The results were analyzed with Rotor-Gene 6000 v1.7 software (Qiagen). A standard curve was constructed for each target gene. Data obtained by amplification with genomic DNA samples could be compared as the amount of genomic DNA was the same for the three T. cruzi isolates. To estimate the copy numbers of each target gene in the three isolates, data were normalized separately using the genome size of each T. cruzi isolate (Souza et al., 2011 (link)). All experiments were performed in triplicate.