Computational protocol: Evidence of endometrial amino acid metabolism and transport modulation by peri-ovulatory endocrine profiles driving uterine receptivity

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Protocol publication

[…] Total RNA was extracted from endometrial samples using the RNeasy Mini Kit (Qiagen Laboratories, Germantown, MD, USA) following manufacturer instructions. The concentration and purity of mRNA were estimated using NanoVue (GE Healthcare Life Sciences, Buckinghamshire, England). Total RNA extracts were stored at −80 °C until cDNA synthesis. One microgram of total RNA was used for reverse transcription using the High-Capacity cDNA Reverse Transcription kit (Life Technologies, Carlsbad, CA, USA), according to manufacturer’s instructions.Quantification of endometrial gene expression was obtained by qPCR analysis using a StepOne Plus® apparatus from Applied Biosystems. Transcript abundance was determined for several AA-transport-related genes. The genes tested were SLC1A1, SLC1A4, SLC1A5, SLC6A1, SLC6A6, SLC6A14, SLC7A2, SLC7A4, SLC7A5, SLC7A7, SLC7A8, SLC7A11, SLC17A5, SLC17A9, SLC36A2, SLC38A1, SLC38A4, SLC38A6, SLC38A7, SLC43A2, SCLY, and DDO. Each primer pair was analyzed, considering the probabilities of hairpin, homodimer and heterodimer formation, using Oligo Analyzer 3.1 software (IDT®; http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/). Sequence specificities were tested using the software Basic Local Alignment Search Tool (Blast) (http://blast.ncbi.nlm.nih.gov). PCR product identity was confirmed by sequencing (Table ). Transcript abundance was compared between tissues from animals in the LF-LCL and SF-SCL groups from Experiment 1 (n = 8 per group) and Experiment 2 (n = 8 for LF-LCL and n = 9 for SF-SCL group). The selection of reference genes was performed using geNorm software (www.qbaseplus.com) []. After selection performed by the software, RPS18 was selected as an endogenous control for endometrium on D4, and cyclophilin, β-actin and GAPDH were chosen for endometrium on D7. Transcript abundance was calculated as the relative abundance between the target gene and the geometric average of selected housekeeping genes and given as an arbitrary value. […]

Pipeline specifications

Software tools OligoAnalyzer, BLASTN
Application qPCR
Organisms Bos taurus
Chemicals Alanine, Amino Acids, Cystathionine, Gonadotropin-Releasing Hormone, Progesterone, Taurine