Abi prism 7900ht fast real time pcr sequence detection system

RNA was extracted from cells by TRIzol reagent (Invitrogen, Carlsbad, CA, USA). All the extracted RNA samples were diluted to 1 μg/μL using nuclease-free water. cDNA was synthesized using 1.0 μL of RNA and 4 μL of qScript cDNA Supermix (Quanta Biosciences, Gaitherburg, MD, USA) in 15 μL of nuclease-free water. Real-time PCR was performed using 1 μL of cDNA, 3.6 μL of nuclease-free water, and 0.2 μL of each forward and reverse primers. Gene-specific primers were used in each reaction; the sequences of the primers were as follows: CYP1A1 sense 5′-GAC CAC AAC CAC CAA GAA C-3′, antisense 5′-AGC GAA GAA TAG GGA TGA AG-3′, and all the results were normalized to β-ACTIN (sense 5′-GGC ATA GAG GTC TTT ACG GAT CTC-3′, antisense 5′-TAT TGG CAA CGA GCG GTT CC-3′). qPCR assays were carried out on an ABI Prism 7900HT Fast Real-Time PCR sequence detection system (Applied Biosystems, Waltham, MA, USA) using SYBR Green PCR Master Mix and analyzed according to the ΔΔCT method.

RNA was extracted from ~35 mg of colon tissue with TRIzol reagent (Invitrogen). RNA extracts were normalized to 500 ng/µL, and 2 µL of RNA was used to synthesize cDNA with qScript cDNA SuperMix (Quanta bio). Then, 1 µL of cDNA was mixed with Fast SYBR Green Master Mix (applied Biosystems) and appropriate primers for occludin (Ocln), zonula occludens 1 (Zo-1), and zonula occludens 2 (Zo-2) (primer sequences are available in Table S4) for qPCR analysis (40 cycles of 95 °C for 20 s, 95 °C for 0.01 s, 60 °C for 20 s, 95 °C for 15 s, 60 °C for 16 s, and 95 °C for 15 s) on the ABI Prism 7900 HT Fast Real-Time PCR sequence detection system (Applied Biosystems). All qPCR results were normalized to Gapdh and analyzed with the ΔΔCT method.

Escherichia coli (wild type [WT] strain MG1655) was cultured in Luria-Bertani medium at 37°C and 220 rpm in the incubator overnight. Spectrophotometer readings at an optical density at 600 nm (OD₆₀₀) were obtained (Eon microplate spectrophotometer; Bio-tek) to estimate bacterial numbers. A series of diluted E. coli media (dilution degree is based on estimated bacterial numbers) were cultured on LB agar plates under the same conditions (24 h at 37°C). All plate cultures were analyzed in triplicate. The colony counts were averaged to determine the total bacterial number, represented as CFU. In parallel, DNA from the same E. coli culture was extracted using the E.Z.N.A. stool DNA kit (Omega Bio-tek). Quantitative PCR assays were carried out using 16S rRNA universal primers (8F, 5′-AGAGTTTGATCCTGGCTCA-3′; 338R, 5′-CTGCTGCCTCCCGTAGGAGT-3′) on serially diluted DNA with Fast SYBR green qPCR master matrix on an ABI Prism 7900HT Fast real-time PCR sequence detection system (Applied Biosystems, Foster City, CA). The reactions were analyzed according to the ΔΔC_T method. qPCR conditions were 95°C for 20 s, 95°C for 3 s, and 60°C for 30 s, for 45 cycles. A standard curve was constructed with the threshold cycle (C_T) value versus the microbial number.

Total RNA was isolated using Trizol (Invitrogen) as per the manufacturer’s protocol. cDNA was synthesized from 2 μg of total RNA using the Superscript III first strand cDNA synthesis kit (Invitrogen) with Oligo (dT) primers. Real Time PCR reactions were done in a 10 μl volume comprising of primer, cDNA template and 1× SYBR Green PCR master mix (Applied Biosystems). GAPC was used as the internal normalization control. PCR was performed on the ABI Prism 7900 HT Fast Real-time PCR Sequence Detection System (Applied Biosystems) in a 384 well reaction plate according to the manufacturer’s recommendations. Primers were F_PANS1qRT and R_PANS1qRT for PANS1 and F_GAPCqRT and R_GAPqRT for GAPC. Cycling parameters consisted of 2 min incubation at 50 °C, 10 min at 95 °C and 40 cycles of 95 °C for 15 s, 57 °C for 30 s and 68 °C for 30 s. Each PCR reaction was performed in three technical replicates across four biological replicates. Specificity of the amplifications was verified at the end of each PCR run using ABI prism dissociation curve analysis. Quantification of mRNA was determined from threshold cycle (Ct values) obtained in the log-linear range of real time PCR amplification plots [38 (link)]. The Mann–Whitney U test performed on mean ΔCt values indicated that the leaf and root samples were significantly different from the inflorescence samples (p < 0.05).