Abi prism 7900 ht sds instrument

Methods for RNA extraction from PMNL and endometrium, primer design and evaluation, cDNA synthesis, quantitative reverse transcription PCR and gene function are presented in the Additional files 2, 3 and 4. Briefly, RNA samples were extracted using Qiazol reagent in combination with the miRNeasy® Mini Kit (Cat. #217004, Qiagen). Thirty-two target genes involved in inflammation response, oxidative stress, eicosanoid metabolism, cellular receptors, transcription regulation and glucose metabolism were evaluated in the PMNL, while 30 target genes related to inflammation, oxidative stress, eicosanoid metabolism, transcription regulation and antimicrobial peptides were assessed in the endometrium. Primers were designed via Primer Express 3.0.1 software (Applied Biosystems). Quantitative PCR (qPCR) was performed in an ABI Prism 7900 HT SDS instrument (Applied Biosystems). Details of primer sequences and amplicon size, primer product sequencing information, and qPCR performance are presented in the Additional file 5, 6, 7 and 8. For PMNL, the internal controls were GOLGA5, SMUG1, and OSBPL2 [17 (link), 18 (link)], while for endometrium were GAPDH, RPS9, and UXT. The geometric mean of the internal control genes was used to normalize the expression data.

Liver was sampled via puncture biopsy [20 (link)] from cows under local anesthesia (i.m. 10 mL lidocaine HCl, 2% soln) at approximately 0800 h on -30, -15, 10, and 30 d relative to parturition. Liver was frozen immediately in liquid nitrogen and stored until further analysis. Specific details of RNA extraction from liver, cDNA synthesis, and quantitative reverse transcription PCR are presented in the supplementary material (S1 File). Briefly, RNA was extracted from samples using Qiazol reagent in combination with the miRNeasy® Mini Kit (Cat. #217004, Qiagen). The quality of RNA evaluated by RNA integrity number in the 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) was 7.2 ± 1.1. Twenty-three target genes involved in inflammation, oxidative stress, fatty acid metabolism, hepatokines, gluconeogenesis, and the methionine cycle were selected. Primers were designed via Primer Express 3.0.1 software (Applied Biosystems).
Quantitative PCR (qPCR) was performed in the ABI Prism 7900 HT SDS instrument (Applied Biosystems). Details of primer sequences, amplicon size, and sequencing results are presented in Tables A and B in S1 File.

Messenger RNA expression of the major RASSF1 variants RASSF1A, RASSF1B and RASSF1C was determined in 14 xenografted PDAC and 8 PDAC cell lines. RNA samples were retrotranscribed to cDNA using the First Strand cDNA Synthesis Kit (Roche). A reverse transcriptase minus cDNA was prepared for each sample as a control. QRT-PCR was carried out as previously described [36 (link)] in 25 μl total volume containing 4 ng of cDNA, 1x Power SYBR Green I Master Mix (Applied Biosystems), 400 nM of each primer. After a starting denaturation for 10 min at 95 °C, 45 PCR cycles (15 s 95 °C and 1 min 60 °C) have been performed on ABI PRISM 7900HT SDS instrument (Applied Biosystems). The relative expression level was calculated using transcript level of RPLPO as reference gene and the standard (=1) was the average of the levels of expression of all samples. QRT-PCR data analysis was performed according to the comparative method following the User Bulletin #2 (Applied Biosystems).