Abi prism 7900ht sequence

Among the 22 cattle samples, five of them were chosen to perform qRT-PCRs on the basis of a large range of total read numbers. These samples showed around 10.10⁶ (1475), 13.10⁶ (1455), 20.10⁶ (1479), 24.10⁶ (1345), and 30.10⁶ reads (1476), respectively. These experiments were conducted using custom-made TLDA (Taq-Man Low Density Array) cards and ABI PRISM 7900HT sequence detector system (Applied Biosystems). The dataset was built with genes involved in glycosylation metabolism, named glyco-genes in the following. They concern glycosyl-transferases, glycosidases, sulfo-transferases, sugar carriers, and lectines. Among the around 800 genes recorded in the bovine genome (unpublished data), 372 were selected according to two criteria: the greatest diversity of the glyco-gene groups and the availability of primers provided by Applied Biosystems (https://bioinfo.appliedbiosystems.com/genome-database/gene-expression.html). Twelve housekeeping genes (18S RNA, TFIID transcription factor, etc., see [17 (link)]) were added as controls to complete the 384-microwells of each microfluidic card. The quantification was done using the SDS 2.3 software (Applied Biosystems) according to the ΔCt method (see the User Bulletin #2 for ABI PRISM 7700 of October 2001). Briefly, ΔCt corresponds to the threshold cycle (Ct) for each gene minus that of the mean of the twelve endogenous internal controls.

RNA was extracted using TRIzol reagent (Life Technologies, Grand Island, NY, USA) and cDNA was synthesized using oligo-dT and random primers (TaKaRa). qPCR was performed using the ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA) with commercial primers generated for the system. β-actin was used as the internal control. Primers were listed in Additional file 1.

Real-time quantitative RT-PCR (RT-qPCR) was performed as described59 (link) using the gene-specific primers designed based on the target sequence (Supplemental Table S10). Total RNA was treated with Turbo DNase (Ambion) to eliminate genomic DNA, and 5 μg of DNase-treated RNA was reverse-transcribed using Superscript III reverse transcriptase (Invitrogen) with oligo d(T)20 primers. The cDNA (1:10) was then used for RT-qPCR. The RT-qPCR was performed using gene-specific primer sets (Supplementary Table S10) and Power SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA) in an optical 384-well plate with an ABI Prism 7900 HT sequence detection system (Applied Biosystems). Melt-curve analysis was performed to monitor primer-dimer formation and to check amplification of gene-specific products. The average threshold cycle (CT) values calculated from triplicate biological samples were used to determine the fold expression relative to the controls. Primers specific for Ubiquitin were used to normalize small differences in template amounts.

Total RNA was extracted using TRIzol reagent method (Invitrogen). Complementary DNA (cDNA) was obtained via reverse transcription of equal amount of RNA following protocols and subsequently mixed with primers, SYBR Green PCR MIX (Applied Biosystems) and germ-free water. The reaction products were run on ABI Prism 7900HT sequence detection system (Applied Biosystems), and serial cDNA dilutions were used to generate GAPDH intensity reference standards according to the comparative Ct method11 (link). The primer sequences of all genes were listed in Table S5.

Total RNA from cells or tumors was isolated using Trizol Reagent (Thermo Fisher Scientific), following the manufacturer’s recommendations. RNA was reverse transcribed by using Applied Biosystem high capacity cDNA reverse transcription kit. Gene expression analysis was performed using TaqMan Gene Expression Assays (Applied Biosystems) on an ABI Prism 7900HT sequence detection system (Applied Biosystems). We used 18S (Thermo Fisher) as the endogenous control throughout all experimental analysis. Analysis was performed using the ^Δ−ΔCt method to determine fold changes. We used gene-specific primers and the Universal Probe Library System (Roche Applied Sciences). Primers are listed in Supplementary Table 4. Cells were treated or not with TNF-α 10 nM 4 h before RNA extraction.