Abi 7500 fast real time system

RNA was isolated from S. lividans strains, and RNA concentration was measured using the NanoDrop 2000 Spectrophotometer (Thermo). Genomic DNA was removed using RNase-free DNase I, and elimination of DNA from RNA was further confirmed using PCR. Reverse transcription was conducted with the cDNA Synthesis Kit (Thermo), and the resulting cDNA was used as the template for quantitative real-time PCR. The expression of dndC, dndD, and dndE was quantitatively analyzed; rrnA encoding 16s rRNA was used as the internal control. Forward and reverse primers are listed in Supplementary Table S2. PCR products were examined by electrophoresis on a 1.2% agarose gel and visualized by staining with ethidium bromide.
For quantitative real-time PCR, reactions were performed on an ABI7500 Fast Real Time System (Applied Biosystems). A reaction mixture of 25 μL contained 1 or 150 ng of cDNA (1 ng for reference gene, 150 ng for target gene), 50 nM forward and reverse primers, and 12.5 μL SYBR Green qPCR Master Mix (Thermo). The conditions for PCR amplification were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 1 min. A dissociation curve ranging from 55 to 90°C in 0.5°C increments, with a dwell time of 30 s, was performed to assess the specificity of the reactions.

The total RNA of cancerous and noncancerous tissues was obtained from patients with HSCC and from FaDu cells by using TRIzol reagent (Invitrogen; Life Technologies, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. The 10-μl reaction volume contained 25 ng cDNA, 0.5 μl mTOR gene expression assay (Hs00234508_ml, Applied Biosystems) and 5 μl 2 × TaqMan Universal PCR Master Mix (Applied Biosystems) and was run in an ABI 7500 Fast Real-Time System (Applied Biosystems). The thermal cycling parameters of PCR were 95 °C for 10 min followed by 40 cycles of 95 °C for 20 s and 60 °C for 1 min. The expression level of the mTOR gene was normalized to the internal control ACTB to obtain the relative threshold cycle (ΔCt). The relative expression in cancerous tissue compared with noncancerous tissue (ΔΔCt) is calculated as follows: ΔΔCt = [ΔCt (cancerous tissue – ACTB) – ΔCt (noncancerous tissue – ACTB)]. The fold change in cancerous tissue compared with untreated noncancerous tissue was then calculated using the 2^−ΔΔCt method.

The Th1 responses were determined by intracellular staining for IFN-γ production by CD8⁺ T cells. The inguinal lymph node cells were isolated from mice with various treatments and stimulated with recombinant HER-2/neu protein (10 μg/mL, R&D System) in complete RPMI 1640 medium overnight. The cells were stained with anti-CD8 and anti-IFN-γ antibodies and then analyzed by flow cytometry. The percentage of CD8⁺IFN-γ⁺ cells in total CD8⁺ T cells is shown in each graph. In addition, the amount of IFN-γ and IL-4 was measured by qPCR. CD4⁺ T cells were purified using the Dynal CD4 Negative Isolation Kit (Dynal AS, Oslo, Norway). Then, total RNA was prepared using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was generated using oligo dT as primer and reverse transcriptase (Promega, Madison, Wisconsin, USA) according to the manufacturer’s instructions. Primer pairs for IFN-γ, IL-4, and hypoxanthine guanine phosphoribosyl transferase 1 (HPRT) were used as described previously [20 (link)]. ABI 7500 Fast Real-Time system with SYBR Green PCR Master Mix (Applied Biosystems) was used for quantitative real-time PCR. The expressions of IFN-γ and IL-4 were normalized with HPRT expression. The data are shown as fold change compared to the vector only control group.

Total genomic DNA was extracted from infected cells via using a commercial viral DNA purification kit (Tiangen, cat# DP-348) following the manufacturer's instructions. Freshly prepared DNA was used as a template for real-time qPCR to measure viral DNA levels with gC-specific primers, as described above.
Analysis of GAPDH was used as an internal control. Real-time PCR was carried out using the ABI 7500 fast real-time system (Applied Biosystems, CA). The levels of viral genome represented by gC were normalized to that of the GAPDH gene. The relative levels of the vial genome were calculated using the method (2^-ΔΔCT) by comparison to the control.

The total RNA was extracted from collected samples using TRIzol RNA isolation reagent (Sigma-Aldrich, MO, USA) following the manufacturer’s instructions for further transcriptome sequencing. For qRT-PCR assay, the isolated total RNA was treated by DNase I to remove the genomic DNA, and cDNA was synthesized using a cDNA Synthesis SuperMix Kit (TransGen Biotech, China). qRT-PCR was performed on ABI 7500 Fast Real-Time System (Applied Biosystems, USA) using a TransStart Top Green qPCR Supermix (TransGen Biotech, China) to verify the transcript levels of target genes involved in quinoa germination, and CqMON gene was used as the internal control to calculate the genes expression by the 2^–ΔΔCt method [42 (link)]. Each qRT-PCR analysis was repeated for three times with three biological replicates, and gene-specific primers were showed in Table S4.

The mature miRNA reverse transcription was performed with miR-specific stem-loop forward primers and a universal reverse primer, URP. These miR-specific primers were designed according to the mature miRNA sequence [73 (link)] (Additional file 13). For real-time PCR, cDNA was mixed with 2 × SYBR Green Mix (Takara, Japan) and each of the miRNA specific primers and universal reverse primer in a final volume of 20 μl. The PCR process was performed using SYBR Green as fluorescence dye and run on 96-wells plates with the ABI 7500 FAST Real Time system (Applied Biosystems). PCR was performed with 95 °C for 30 s, 40 cycles of 95 °C for 3 s, 60 °C for 30 s and 72 °C for 30 s. Ubiquitin 7 (GhUBQ7) (Additional file 13) was used as the reference gene [74 (link)]. The relative expression level of miRNA was calculated by 2^-△△Ct. Three biological replicates were performed.

Isolation of total peripheral blood leukocytes, RNA extraction, cDNA synthesis, and qRT-PCR were performed as previously described (17 (link)). Briefly, the 2 μg RNA input for cDNA synthesis was determined by spectrophotometric OD260 measurement, and cDNA was generated with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), following the manufacture's protocols. Expression of the NR1D1 and RORC genes was analyzed using the TaqMan system, and all TaqMan Gene Expression Assays were purchased from Applied Biosystems. Expression of the human housekeeping gene, ACTB (actin beta), was used for normalizing NR1D1 and RORC expression in real-time qRT-PCR. Real-time qRT-PCR was performed in an ABI 7500 Fast Real-Time System (Applied Biosystems). The expression levels of the NR1D1, RORC, and BMAL1 were normalized to the internal control ACTB to obtain the relative threshold cycle (ΔCt).