Computational protocol: Ontogenetic Expression of Lpin2 and Lpin3 Genes and Their Associations with Traits in Two Breeds of Chinese Fat-tailed Sheep

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Protocol publication

[…] Total RNA was isolated using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and was purified with the RNeasy Mini Kit (QIAGEN GmbH, Hilden Germany) as recommended by the manufacturers’manual. Single-stranded cDNA was synthesized from one microliter of total RNA using the PrimeScript RT reagent Kit (Takara, Biotechnology, Dalian, China), according to the manufacturer’s guides. Target and housekeeping genes’ mRNA expression was carried out by Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Foster, CA, USA) and SYBR Premix Ex Taq II kit (Takara, Biotechnology, China) following the manufacturer’s instructions. Lpin2 and Lpin3 mRNA expression was normalized to housekeeping genes RPL13A and B2M mRNA levels respectively, data analysis was performed using the comparative CT method (), and the final relative expression was obtained by geometric mean. Primers () were designed according to the sheep mRNA sequence (GenBank accession number XM_004020631 and XM_012155543.1) and those for sheep B2M and RPL13A by using online Primer3 plus ( PCR amplification procedure was as follows: 95°C for 30 s, 42 cycles at 95°C for 5 s and 61°C for 35 s, and a following cycle at 95°C for 15 s, 61°C for 1 min and 95°C for 15 s to obtain the dissociation curves. The standard curves were derived from a 2-fold dilution step by step with eight levels for target and housekeeping genes. PCR efficiencies of the four genes were between 90% and 110%. The R2 values of standard curves ranged from 0.974 to 0.998, and melting curves showed a single peak. Relative ratios of Lpin2 or Lpin3 to RPL13A and B2M calculated using the standard curves confirmed that the real-time PCR results were trustworthy. All the reactions were executed in triplicate. [...] The relative abundance of Lpin2 and Lpin3 mRNA was analyzed by using general linear model in SPSS Statistics 19.0 software package (SPSS Science, Chicago, IL, USA). Four factors including breed, sex, age, tissue and their two-way interactions were considered as the influencing factors on the mRNA expression. The model was fitted as the following function:where, yijklm is the mRNA expression amount, μ the overall mean, Bi the ith breed (i = 1, 2), Sj the jth sex (j = 1, 2), Tk the kth tissue (k = 1, 2, ..., 8), Ml the lth months of age (l = 2, 4, ..., 12), BSij, BTik, BMil, STjk, SMjl, and TMkl correspond to the two-way interactions, and eijklm is the random residue. Multiple comparison among levels within different factors were performed by the Duncan’s method and the significance level was set at (p<0.05). The Pearson correlation coefficients between mRNA expression and traits were obtained by using SPSS Statistics 19.0 software package (SPSS Science, Chicago, IL, USA). […]

Pipeline specifications

Software tools Primer3, SPSS
Applications Miscellaneous, qPCR
Organisms Ovis aries, Homo sapiens