7500 real time rt pcr system

RNA was extracted from the different samples
of iPSC and ES cell aggregates, at days 8 and 14
of differentiation using TRIzol Reagent (Invitrogen,
USA). Total RNA was treated with DNase I
to remove genomic DNA contamination. Two micrograms
of total RNA was used for the reverse
transcription reaction with the first strand cDNA
synthesis kit (fermentas, UK) and random hexamer
primer, following manufacture’s instruction.
Quantitative polymerase chain reactions (PCR)
were set up in three biological replications with the
Power SYBR GreenMaster Mix (Applied Biosystems,
USA) and analyzed with the 7500 real-time
(RT) PCR system (Applied Biosystems, USA).
Expression values were normalized to the average
expression of the housekeeping gene Glyceraldehyde
3-phosphate dehydrogenase (GAPDH). The
primer sequences are presented in Table 1.

Total RNA was isolated from the tissue samples using the RNeasy kit (Qiagen). After that, mRNA was reverse-transcribed into cDNA using the oligo-dT primer (Roche). Quantitative real-time RT-PCR was then carried out using Applied Biosystems 7500 real-time RT-PCR system (Applied Biosystems, Foster City, CA) using TaqMan gene expression assays (Applied Biosystems) for LH1 (assay ID Hs00609368_m1), LH2 (assay ID Hs00168688_m1), LH3 (assay ID Hs01126612_m1), LOX (assay ID Hs00184700_m1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (assay ID Hs99999905_m1). The amount of RT-PCR product for the gene of interest was normalized to the quantity of GAPDH. Each assay was performed in triplicate.

The SFTSV gene in cat samples was detected using real-time RT-PCR, described previously [26 (link)]. RNA was extracted using Isogen-LS (Nippon Gene, Tokyo, Japan), and the RT-PCR reaction was performed using a One-Step PrimeScript RT-PCR Kit (Takara Bio Inc. Shiga, Japan) and 7500 Real-time RT-PCR System (Applied Biosystems, Massachusetts, MA, USA). SFTSV-specific primers and a probe were designed based on a consensus sequence of the RdRp gene [26 (link)]. The copy numbers were calculated as the ratio of the copy numbers to the standard control prepared from a cloned plasmid vector [26 (link)].

Real-time PCR was carried out on Applied Biosystems 7500 Real-Time RT-PCR System with the following program: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. All cDNA samples were tested three times, and the results were normalized to the levels of GAPDH expression using the SYBR Premix Ex Taq II system (Takara, Japan). Based on the mRNA sequences of rabbit PGRPs published in GenBank, real-time PCR primers for PGLYRP-1, PGLYRP-2, and PGLYRP-3 were designed and are shown in Table 1. The following equation was used to obtain the fold change: $RQ=\frac{{\left(1+E_a\right)}^{C{T}_{ca}-C{T}_{ta}}}{{\left(1+E_k\right)}^{C{T}_{ck}-C{T}_{tk}}}$ , where E_a is the amplification efficiency of the target gene, E_k is the amplification efficiency of the internal control gene, CT_ca is the C_t value of the target gene in the control group, CT_ta is the C_t value of the target gene in the test group, CT_ck is the C_t value of the internal control gene in the control group, and CT_tk is the C_t value of the internal control gene in the test group.

The total RNA was extracted from the lateral roots and galls using RNeasy Plant kit (Huayueyang, Beijing, China) according to the manufacturer’s protocol. For each case, 1 μg RNA was used for cDNA synthesis using the Faster Quant RT kit (Qiagen, Beijing, China). The cDNA samples were used as templates for RT-qPCR analysis. Quantitative RT-PCR was performed in 384 well plates with a 7,500 real-time (RT) PCR System from Applied Biosystems using SYBR Green according to the manufacturer’s protocol (Takara, Japan). All of the samples were tested in five biological replicates with two technical replicates for each reaction. CsUBQ (Csa4G089780) and CsEF1α (Csa2G139820) were employed as internal reference genes. The results were determined by using the 2^–△△CT method (Schmittgen and Livak, 2008 (link)). The primers and gene accession data are listed in Supplementary Tables 1, 2, respectively.

qPCR was performed using the Fast Start Universal SYBR Green Master Mix (Applied Biosystems, Foster City, CA). RT-PCR was performed using quantitative PCR systems (Applied Biosystems® 7500 Real-Time PCR Systems, Thermo Fisher Scientific, Waltham, MA, USA) with corresponding primers (Table S1, Invitrogen). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was simultaneously assayed as a loading control. The expression levels of mRNA were reported as fold changes vs. sham control. Reactions were performed using an Applied Biosystems 7500 Real-Time RT-PCR System. Data was analyzed using a ΔΔCT approach.