Quantstudio real time pcr software v1

Isolation of total RNA and reverse transcription into cDNA was performed as described recently [3 (link)]. Relative quantitative gene expression was measured via real-time PCR using a QuantStudio 6 Flex Real-Time PCR System with QuantStudio Real-Time PCR Software v1.3 (Applied Biosystems, Foster City, CA, USA) Target gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression as internal standard and calculated as fold induction in comparison to untreated controls. All primer sequences used for qPCR are given in Table A1.

The five broilers of each line from the same generation of the NEAUHLF were selected randomly to perform the validate experiment. The total RNA of abdominal fat tissue was extracted using TRizol reagent (Invitrogen, CA, USA) following the manufacturer’s protocol. According to the recommended protocol of the PrimeScript™ RT reagent kit (Takara Bio Inc., Kusatsu, Japan), the probable genomic DNA in isolated RNA was firstly removed by gDNA Eraser followed by the reverse transcription reaction. Specific primers (Additional file 1: Table S11) for candidate RNAs (three each of the DE lncRNAs and DE circRNAs were randomly selected) were designed using Primer premier 5.0 software. The ABI QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was adopted to perform the qRT-PCR of candidate genes with FastStart SYBR Green Master (Roche, Mannheim, DEU). The reaction conditions were: an initial denaturation at 95 °C for 10 min, 40 cycles of 15 s at 95 °C and 1 min at 60 °C. C_t values of all genes were analyzed using QuantStudio™ Real-Time PCR Software v1.3 (Applied Biosystems). The housekeeping gene of TBP was used for normalization of C_t values. Gene expression comparison between the two lines was done using the 2^−ΔΔCt method.

Total RNA was extracted using either the RNeasy Mini kit (Qiagen) or Arcturus PicoPure RNA isolation kit (Applied Biosystems), and reverse transcription was performed with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturers’ instructions. qPCR was performed with the SYBR Green gene expression assay (Applied Biosystems) using the ViiA 7 Real-Time PCR System (Applied Biosystems) and analyzed with Quant Studio Real-Time PCR software v.1.3 (Applied Biosystems). Primer pairs are listed in Supplementary Table 11.

To assess mRNA expression, total RNA was isolated from cells and tissues then subjected to cDNA synthesis^{29 (link)}. Quantitative real-time PCR (qPCR) reactions were set up with Power SYBR Green PCR Master Mix and were run with default cycle parameters on the Applied Biosystems 7500 Fast Real-Time PCR System (for 96-well plate format) or the Applied Biosystems ViiA7 Real-Time PCR System (for 384-well plate format). Applied Biosystems 7500 software v2.3 and Applied Biosystems QuantStudio Real-Time PCR software v1.3 were used for qPCR data analysis. Gene expression was quantified using the 2^−ΔΔCT method and relative mRNA expression was normalised to 18S rRNA levels which have been shown to display consistent expression across the cells and conditions studied. All qPCR primers were designed using primer3 (http://primer3.ut.ee/) and can be found in Supplementary Table 2. For microarrays on eosinophils sorted from subcut SVF by FACS, RNA was isolated using the RNeasy Micro Kit (Qiagen) then subjected to quality control using an Agilent 2100 Bioanalyzer, following preparation with an Agilent RNA 6000 Pico Kit. An Affymetrix 3’ IVT Pico Kit was used prior to microarrays which were performed on an Affymetrix GeneChip HT MG-430PM Array Plate. Partek Genomics Suite v7 software was used for data analysis, and microarray datasets are available from GEO (Accession No. GSE117445).

Whole blood was collected in PAXgene Blood RNA (PreAnalytiX) or Tempus Blood RNA (Applied Biosystems) Tubes (N = 16 and 47, respectively) [40 ]. Total RNA including miRNA was extracted using the PAXgene Blood miRNA Kit (Qiagen), or the Tempus™ Spin RNA Isolation Kit (Applied Biosystems), respectively, following the manufacturer´s protocols. The concentration of the RNA samples and the sample purity was assessed with NanoDrop 1000 Spectralphotometer (Thermo Scientific). The cDNA was synthesized by a reversed transcription reaction using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative PCR was performed on the QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems by Life Technologies) by using TaqMan Fast Advanced Mastermix (Applied Biosystems), and the MECP2 TaqMan Gene Expression Assay Hs00172845_m1 (Applied Biosystems). The Actin Beta (ACTB) TaqMan Gene Expression Assay Hs01060665_g1 (Applied Biosystems) was used as an internal standard. Results were calculated with the QuantStudio Real-Time PCR Software v1.3 (Applied Biosystems by Thermo Fisher Scientific) by the comparative 2^−ΔΔCt method and normalized to ACTB. Analyses were carried out in triplicates. An unpaired t-test revealed that blood tubes did not significantly influence MECP2 levels (t₆₁ = −0.534, p = 0.595).

Nasopharyngeal swabs were tested for SARS-CoV-2 RNA using the CDC 2019-nCoV RNA RT-PCR diagnostic panel (Integrated DNA Technologies, Iowa, USA). A cycle threshold (Ct) value of <40 was considered positive for SARS-CoV-2 using QuantStudio Real-Time PCR software v1.3 (Applied Biosystems, UK). Ribonuclease protein was used as an internal control to identify presence of human RNA. A negative extraction control and a PCR no-template control were also performed with every test. The results of patients with positive PCR tests were shared with the Blantyre District Health Office for further follow up and patient management.