Taqman universal pcr kit

Total RNA was extracted from lymphocytes and retinal cells using the TRIzol reagent according to the procedures recommended by the manufacturer (Life Technologies, Gaithersburg, MD). All RNA samples were digested with RNAse-free DNAse 1 (Life Technologies) for 30 min, purified by phenol/chloroform extractions, and precipitated in 0.4 M LiCl. RNA (10 µg), a commercial synthesis system (SuperScript III Reverse Transcriptase; Life Technologies), and oligo(dT) were used for first-strand synthesis as previously described. First-strand synthesis containing each mRNA sample but without reverse transcriptase was performed to control for possible DNA contamination; failure to obtain real-time PCR (RT-PCR) products with any of the PCR amplimers confirmed the absence of contaminating DNA. All cDNA preparations used were suitable for PCR amplification on the basis of efficient amplification of a β-actin sequence. RT-PCR was performed on a fast RT-PCR system (ABI 7500) and PCR parameters were as recommended by the manufacturer (TaqMan Universal PCR Kit; Applied Biosystems). Primers and probes were from Applied Biosystems: Il12a (ABI, Mm00434169_m1), Ebi3 (ABI, Mm00469264_g), Il10 (ABI, Mm00439616_m1), gapdh (ABI, Mm99999915_g1), actin b (Mm00469264_g1) (Supplementary Table 1). The mRNA expression levels were normalized to the levels of GAPDH housekeeping gene.

SAS-L1 cells were transiently transfected with 75 pmol EFNB2 siRNA and cultured for 24 h. The cells were then cultured in the presence of 2 μg/mL clustered Fc, ephrin-B2/Fc or Eph-B4/Fc for 24 h. Total cellular RNA was extracted using the RNeasy total RNA isolation system (Qiagen, Valencia, CA). The extracted RNA (1 μg) was added to 20 μL reverse transcription buffer (50 mM Tris-HCl pH 8.3, 50 mM KCl, 10 mM MgCl₂, 1 mM EDTA, 10 μg/mL bovine serum albumin, and 1 mM dithiothreitol [DTT]) containing 10 mM dNTP, 50 U RNase inhibitor, 1μg oligo dT-primer, and 50 U avian myeloblastosis virus reverse transcriptase (all from Takara Biomedicals, Kyoto, Japan). The reaction mixture was incubated at 42°C for 40 min and heated at 99°C for 5 min. Quantitative real-time reverse transcription-polymerase chain reaction was performed using a master mix provided in the TaqMan universal PCR kit and the ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA). Primers for EFNB2 mRNA and the TaqMan probes were obtained from Applied Biosystems. Expression level of the endogenous reference gene human β-actin was determined using the commercially available TaqMan predeveloped assay reagent (Applied Biosystems).

Total RNA was extracted using the Trizol reagent according to the procedures recommended by the manufacturer (Life Technologies, Gaithersburg, MD). RNA integrity was verified by analysis of 18S and 28S ribosomal RNA expression on RNA gels. RNA (10 μg), SuperScript III Reverse Transcriptase (Life Technologies, Gaithersburg, MD), and oligo (dT)_12–16 were used for first-strand synthesis as previously described [17 (link)]. All cDNA preparations used were suitable substrates for PCR amplification on the basis of efficient amplification of a β-actin sequence and each gene-specific primer pair used for RT-PCR analysis spans at least an intron. Real-time PCR was performed on an ABI 7500 (Applied Biosystems) and PCR parameters were as recommended for the TaqMan Universal PCR kit (Applied Biosystems). Primers and probes were purchased from (Applied Biosystems). The relative mRNA expression levels were normalized to the levels of the housekeeping gene, β-actin, or gapdh. Genomic DNA was isolated from the TG using Wizard Genomic DNA Purification Kit (Promega, WI, USA). DNA specific to the glycoprotein H (gH) gene of HSV-1 was amplified using the RT-SYBR Green ROX qPCR mastermix was purchased (QIAGEN, MD, USA). The qPCR primer sequences are 5’-CGAACAGAGAGCTTGGCATTC-3’ and 5’-TGAGGTAGGTTAGGAACATG-3’.