7000 real time pcr system

RNA extraction, reverse transcription and quantitative RT-PCR were performed as described previously [29 (link)]. Total RNA was extracted with RNeasy mini kit (Qiagen) and reverse transcription was performed with High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) following the manufacturer’s instruction. Quantitative Taqman RT-PCR was performed in triplicate with 7000 Real-time PCR system (Applied Biosystems, Themo Fisher Scientific). GDF-15: Hs00171132_m1, NQO1: Hs01045993_g1, GCLM: Hs00978072_m1 and HMOX1: Hs01110250_m1 probes were purchased from Applied Biosystems. The 2^ΔΔCt method was used to quantify the relative transcriptional level of each gene. Endogenous GAPDH was used as housekeeping gene.

We isolated RNA from the lungs via Triazol (Invitrogen) and treated it with DNase I (Invitrogen). We used random primers (Invitrogen) and Superscript II (Invitrogen) to synthesize first-strand complementary DNAs from equivalent amounts of RNA from each sample. We performed real-time RT-PCR in a 7000 Real-Time PCR System (Applied Biosystems) with SYBR Green PCR Master Mix (Applied Biosystems). Data were generated by the comparative threshold cycle (ΔCT) method by normalizing to HPRT [26 (link)]. Forward and reverse primers amplifying are as follows, respectively:
M2:5′GAGGTCGAAACG CCT 3′ & 5′CTGTTCCTTTCGATATTCTTCCC3′, CYP4F18:5′ AGAGCCTGGTGCGAACCTT 3′ & 5’ TGGAATATGCGGATGACTGG 3’, CYP4F16:5’GGAGTGGCTTCCTGGATTTT3’& 5’ATGCAGGGTCAACAATCCTC3’, TBXAS1:5’AGGCTTCTGAAAGAGGTGGACCT3′ & 5′TGAAATCACCATGTCCAGATAC3′, ALOX5:5′ATGCCCTCCTACACTGTCAC3′ & 5′CCACTCCATCCATCTATACT3′, ALOX5ap:5’CTCCCAGATAGCCGACAAAG3’ & 5’CAGAACTGCGTAGATGCGTA3’, COTL1:5’GATGAGGGCAAACTTGGATCT3’ & 5’GAGCAGATTACCAGCACTTCA3’, GGGT1: 5’AGGAGAGACGGTGACT3' & 5' GGCATAGGCAAACCGA3', DPEP2: 5’CTGACCTTTCTCTGCCACA3’ &5’GAATCTTCCTGATGACCTCCTG3’

The expression pattern of miR-130b was analyzed in cDNA samples obtained from granulosa cells, immature and matures oocytes and their corresponding cumulus cells, and preimplantation embryos using quantitative real time PCR (qPCR). For this, mature miRNA-130b specific primers were purchased from Qiagen (Hilden, Germany). The qPCR was performed by mixing 2.5 μl template cDNA with 12.5 μl of SYBRGreen mix (Qiagen, Valencia, CA), 10× miScript Universal Primer and 10× miScript Primer assay in 25 μl of final volume. The qPCR was performed for 45 cycles of 95 °C for 15 s and 60 °C for 1 min in 7000 Real-Time PCR system (Applied Biosystems, USA). The threshold cycle (C_t) values of miR-130b and the endogenous controls were recorded using Sequence Detection Software (SDS v1.2.1, Applied Biosystems, USA). The qPCR data were normalized using the geometric mean of U6 and small nuclear RNAs (Snord48). The miRNA expression levels were then determined from the triplicate runs using the 2^-ΔΔCt method. All experiments were performed at least in three biological triplicates.