Applied biosystems stepone real time pcr system

Specific transcript levels were determined using quantitative RT-PCR (qPCR). Total RNA was isolated from log phase cultures using RNeasy Mini kit (Qiagen, Valencia, CA). RNA was treated with DNAse1 on-column using RNase-Free DNase Set (Qiagen) and cDNA synthesized using iScript cDNA synthesis kit (BioRad, Hercules, CA). qPCR was performed using diluted cDNAs and Power SYBR green PCR Master Mix (Life Technologies, Carlsbad, CA) with oligonucleotide pairs specifically targeting transcripts for native E6^N and E7^N, and RNAi^R E6^R and E7^R. The positions of these primers are indicated in the sequence alignment presented in S1 Fig. TbZFP3 (Tb927.3.720, nts 241–301) was used as the control amplicon. Amplification was performed using an Applied Biosystems StepOne Real-Time PCR System (Life Technologies, Carlsbad, CA). For each transcript post-amplification melting curves indicated a single dominant product. All calculations and normalizations were done using StepOne software, version 2.2.2. Reactions were performed in technical triplicates, and means ± standard errors of the means (SEM) for three biological replicates are presented.

Total RNA was isolated with the RNAeasy Mini kit (QIAGEN). One µg of purified RNA was reverse transcribed using SuperScript IV VILO master mix (Invitrogen) following the manufacturer’s protocol. Quantitative PCR was carried out using the QuantiFast SYBR Green PCR kit (Qiagen) and analyzed on an Applied Biosystems StepOne Real-Time PCR System (Life Technologies). Each individual biological sample was qPCR-amplified in technical triplicate and normalized to GAPDH as an internal control. Relative quantification was calculated according to the ΔΔCt relative quantification method. Error bars indicate ± SD of three replicates. Primer sequences are available on request.

The dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/) of the National Center for Biotechnology Information and the HapMap database were used to search for single nucleotide polymorphisms (SNPs). Four SNPs (MT1A: rs11640851 and rs8052394; MT2A: rs10636 and rs28366003) were analyzed. We used TaqMan allelic discrimination assays (Applied Biosystems, Foster City, CA, USA) to genotype the SNPs, and the results were read with an Applied Biosystem 7300 Real-Time PCR System (Life Technologies Corp., Carlsbad, CA, USA). In brief, the genotyping of the MT1A and MT2A polymorphisms (rs11640851, rs8052394, rs10636, and rs28366003) was performed via TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA, USA) [41 (link)]. The final volume for each reaction was 10 μL, containing 5 μL of TaqMan Universal PCR Master Mix, 0.25 μL of primers/TaqMan probe mix, and 10 ng of genomic DNA. The real-time PCR comprised an initial denaturation step at 95 °C for 10 min, followed by 40 cycles each at 92 °C for 15 s and 60 °C for 1 min. Fluorescence was measured with an Applied Biosystems StepOne Real-Time PCR System (Life Technologies Corp., Carlsbad, CA, USA). The allele frequencies were determined using ABI SDS software. Genotyping was repeated on a random 10% sample to confirm the results of the original analysis.

Total RNA was extracted using NucleoSpin RNA (Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol. RNA concentrations were calculated from the optical density at 260 nm, assuming an OD260 unit is equivalent to 40 μg/mL RNA. The RNA purity was determined by measuring the absorbance ratio at 260/280 nm. Only samples with a ratio > 1.8 were used. Aliquots of 240 ng of total RNA were reverse transcribed using ReverTra Ace (Toyobo, Osaka, Japan).
qRT-PCR was performed using SYBR Premix Ex Taq II (Takara Bio) on an Applied Biosystems StepOne Real-Time PCR System (Life Technologies) according to the method described previously [35 ]. In brief, the qPCR mixture consisted of 10 μL SYBR Premix Ex Taq II, 0.4 μM forward and reverse primers, 0.4 μL ROX Reference Dye, 2 μL template, and ddH₂O to a total volume of 20 μL. The amplification parameters were as follows: initial denaturation at 95°C for 30 s followed by 50 cycles of denaturation at 95°C for 5 s, annealing and extension at 60–68°C for 34 s, and a melting curve from 60 to 95°C, increasing in increments of 0.5°C every 5 s. Normalization was performed using the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a control. Primer sequences are listed in Table 1. Relative mRNA expression was calculated by the 2^–ΔΔct method. Samples from 5 cows were measured in duplicate.