Viia 7 real time pcr system

Total lung RNA was extracted from homogenized lysates using the mirVana miRNA Isolation Kit (Invitrogen cat. AM1560) following the manufacturer’s protocol for total RNA isolation. Total RNA samples were quantified using a Nanodrop spectrophotometer (Thermo cat. ND-2000C), and reverse transcribed to cDNA using the SuperScript IV VILO Master Mix with ezDNase enzyme (Invitrogen cat. 11766050) following the manufacturer’s instructions. Expression of mouse Ace2, mouse Gapdh, and human ACE2 were quantified by qPCR using the ViiA7 Real-Time PCR System (Thermo cat. 44535545). TaqMan probes targeting mouse Gapdh (Thermo probe ID Mm99999915_g1) and human ACE2 (Thermo probe ID Hs01085333_m1) were used in conjunction with the TaqMan Fast Advanced Master Mix (Thermo cat. 4444556) and the ViiA7 Real-Time PCR System with QuantStudio Software v1.7.2 (Thermo cat. 44535545). Samples were run in technical triplicate in 10ul reaction volumes on a 384-well plate, and hACE2 transcript abundance values were normalized to mGapdh in each sample and relative expression was calculated using a reference lung sample (K18-hACE2) and the delta delta-Ct method.

Eight single-nucleotide polymorphisms (SNPs) (C1584G, C100T, C1023T, G1846A, C2580T, G2988A, G3183A, and G4180C) and one deletion (2615_2617delAAG) in the CYP2D6 gene and the copy number of the gene were assayed by qPCR. We used specific hydrolysis probes for each polymorphism (TaqMan SNP genotyping assays; Thermo Fisher Scientific). All amplification reactions were performed according to the protocol described by Silvino and colleagues (15 (link)). Amplification and fluorescence detection were carried out using the ViiA 7 real-time PCR system (Thermo Fisher Scientific). The results were analyzed by QuantStudio real-time PCR software (Thermo Fisher Scientific) and the Thermo Fisher cloud platform (Thermo Fisher Scientific).
The copy number of the CYP2D6 gene was determined by quantitative PCR (qPCR) using the Hs00010001_cn assay (Thermo Fisher Scientific) for gene deletion and amplification detection. All amplification reactions were performed according to the protocol described by Silvino and colleagues (15 (link)). Amplification and fluorescence detection were carried out in the ViiA 7 real-time PCR system (Thermo Fisher Scientific), and the number of copies was estimated in CopyCaller v.2.0 software. Only samples with confidence values greater than 95% and absolute z-scores of <1.75 were considered in our analysis.

Total RNA was isolated with Trizol reagent (Invitrogen) following RNA purification using the RNeasy Mini Kit (Qiagen) and converted to cDNA by High Capacity RNA-to-cDNA Kit (Applied Biosystems), according to the manufacturer's protocols. An assay comprising known human and murine CLEC16A RNA transcripts as well as control genes (β-Actin and HRPT1) were measured by real time PCR on a ViiA™ 7 Real Time PCR System, using predesigned 20X FAM-MGB TaqMan gene expression assays available from Applied Biosystems. All assays had primers covering exon-exon boarders to avoid DNA contamination. Triplicates were used for all samples included in the experiment. All PCR runs were performed on ViiA™ 7 Real Time PCR System using ViiA7 RUO software v1.2.2 (Life Technologies).

RNA samples were reverse transcribed using 5 ng/μl random primers, 1 U/μl RNase inhibitor (RNAse Out, Invitrogen) and 5 U/μl reverse transcriptase (SuperScript III, Invitrogen), according to manufacturer's instruction. NRIR expression was quantified in duplicates by RT-qPCR from 9 ng RNA-equivalent cDNA in the presence of SYBR Select Master Mix (ThermoFisher Scientific, Applied Biosystems) and 400 nM specific primers (Table S1), on the ViiA™ 7 Real-Time PCR System (ThermoFisher Scientific, Applied Biosystems) using the standard protocol. PCG expression was quantified in duplicates by RT-qPCR from 9 ng RNA-equivalent cDNA in the presence of Fast SYBR Green Master mix (ThermoFisher Scientific, Applied Biosystems) and 200 nM of specific primer pairs (Table S1), on the ViiA™ 7 Real-Time PCR System (ThermoFisher Scientific, Applied Biosystems). Primers were designed using the Oligo Explorer software², for only fifty-six out seventy-nine NRIR putative target genes was possible to design specific primer pairs. Data were analyzed with LinReg PCR 7.0³ and Q-Gene software⁴ Gene expression was calculated as mean normalized expression [MNE (44 (link))] units after normalization over the stably expressed RPL32 or ACTIN B.

To perform HRM SNP barcoding, we first quantified the concentration of DNA for all clinical samples, as described above using a Nanodrop. We then diluted the samples in 1X TE buffer to a concentration of total DNA (human and parasite DNA) at 1 ng/μl based on based on OD₂₆₀. For all assays, we included sequenced control samples in each assay plate to identify the reference and alternate SNP temperature melt (T_m) curves. Next, we prepared a master mix (described above) for each assay. We calculated the amount of master mix by multiplying the volume of each component by the number of PCR wells or test samples. We added 7 μl of the master mix to each well in the PCR plate. We gently vortexed and centrifuged the reaction mixture, then added 3 μl of the DNA dilution at 1 ng/μl (final assay concentration 3 ng/μl) to each well for a total reaction volume of 10 μl. We placed a seal on top of the PCR plate with a roller and then centrifuged at 1000 RPM for 1 min. We optimized this protocol to be performed in either a 48-well plate in the Eco Real-Time PCR System or in a 384-well plate in the Applied Biosystems ViiA 7 Real-Time PCR System. We used HRM software on the Eco Real-Time PCR System or the Applied Biosystems ViiA 7 Real-Time PCR System for HRM genotyping analyses. See supplemental materials S1 File for the full barcode assay protocol.

RNA was reverse transcribed to cDNA using the High Capacity RNA to cDNA kit (Applied Biosystems, Foster City, CA). cDNA was then analyzed by quantitative RT-PCR. Real-Time PCR on microbial DNA was performed using primers found in Supplementary Table 1. Real-time PCR was performed using FastStart Universal SYBR Green Master (Rox) (Roche) and analyzed with ViiA™7 Real-Time PCR System (Applied Biosystems).
Real-time PCR was performed using FastStart Universal SYBR Green Master (Rox) (Roche) and analyzed with ViiA™7 Real-Time PCR System (Applied biosystems).

As described in our previous publication^{37 (link)}, total RNA was isolated with Trizol reagent (Invitrogen) following RNA purification using the RNeasy Mini Kit (Qiagen) and converted to cDNA by High Capacity RNA-to-cDNA Kit (Applied Biosystems), according to the manufacturer’s protocols. Assay comprising known human and murine CLEC16A RNA transcripts as well as control genes (β-actin and HRPT1) were measured by real time PCR on a ViiA 7 Real Time PCR System using predesigned 20X FAM-MGB TaqMan gene expression assays available from Applied Biosystems. All assays had primers covering exon-exon boarders to avoid DNA contamination. Triplicates were used for all samples included in the experiment. All PCR runs were performed on ViiA 7 Real Time PCR System using ViiA7 RUO software v1.2.2 (Life Technologies).