Marker Lnc-482286 related to porcine skeletal muscle satellite cell proliferation, and application of marker Lnc-482286
A technology of lnc-482286 and satellite cells, applied in the field of molecular biology, can solve the problem of less research on porcine tissue-specific LncRNA
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Embodiment 1
[0023] Validation of porcine long non-coding RNA Lnc-482286
[0024] Samples: Longissimus dorsi muscle tissue from Jinfen white pigs that were 1 day old was collected, wrapped in tinfoil paper, quickly frozen in liquid nitrogen, and stored at -80°C.
[0025] Reagent: Reagent was purchased from Life Technologies; PrimeScript RT reagentKit with gDNA Eraser and DNA marker were purchased from TaKaRa; 2×Es Taq Master Mix (Dye) was purchased from Kangwei Century Biotechnology Co., Ltd.; 50×TAE buffer, nucleic acid dye, Triton X-100 and DAPI staining solution were purchased from Suleibao Company; agarose was purchased from Shanghai Sangon Bioengineering Co., Ltd.
[0026] 1) Extraction of total RNA
[0027] Take out the longissimus dorsi muscle tissue of Jinfen white pig 1 day old from the -80°C refrigerator, add 1mL Trizol reagent, grind it fully with a tissue homogenizer, grind and pulverize it, let it stand for 10min, and fully lyse it at 4°C, 12000r· min -1 Centrifuge for 10...
Embodiment 2
[0037] Temporal expression properties of Lnc-482286
[0038] Samples: Longissimus dorsi muscle tissues of Jinfen white pigs were collected for 1 day, 90 days and 180 days respectively, wrapped in tinfoil paper, frozen in liquid nitrogen immediately, and stored at -80°C.
[0039] Reagent: Reagent was purchased from Life Technologies; PrimeScript RT reagent Kit with gDNA Eraser and 2×SYBR Premix Ex Taq II were purchased from TaKaRa.
[0040] According to the sequence (SEQ ID NO:1) of the Lnc-482286 transcript, the Lnc-482286 quantitative primer pair was designed by Oligo7 software, as follows:
[0041] Quantitative primer pair for Lnc-482286:
[0042] Upstream primer: 5'-AACAGGTGAGCAGGATGCGAAG-3' (SEQ ID NO: 4)
[0043] Downstream primer: 5'-GATCACAGTGCCCGTCCTTCAG-3' (SEQ ID NO:5)
[0044] Quantitative primer pairs for 18S rRNA:
[0045] Upstream primer: 5'-CCCACGGAATCGAGAAAGAG-3' (SEQ ID NO: 6)
[0046] Downstream primer: 5'-TTGACGGAAGGGCACCA-3' (SEQ ID NO: 7)
[0047] ...
Embodiment 3
[0051] The role of Lnc-482286 in the proliferation of porcine skeletal muscle satellite cells
[0052] Cells: Porcine skeletal muscle satellite cells were isolated and cultured in the laboratory.
[0053] Reagents: fetal bovine serum (FBS), DME / F12 were purchased from Gibco; penicillin-streptomycin mixture, glycine, 4% paraformaldehyde were purchased from Solebol; Triton X-100 was purchased from Sigma; 0.25% pancreatic Enzymes and PBS buffer were purchased from Hyclon; EdU proliferation detection kit and transfection reagent ribo FECT TM CP was purchased from Guangzhou Ruibo Biotechnology Co., Ltd.
[0054] For the Lnc-482286 sequence, synthesize an siRNA sequence (Smart Silencer) that inhibits its expression. SmartSilencer is a mixture, and the specific sequence is:
[0055] 5'-CGACATGCTCTCTTGGGTCT-3' (SEQ ID NO: 8)
[0056] 5'-CACGTGCCAGGAGACTTGTA-3' (SEQ ID NO:9)
[0057] 5'-CAGAACAGGTGAGCAGGAT-3' (SEQ ID NO: 10)
[0058] 5'-TCTTGTTTCCATGGTGACT-3' (SEQ ID NO: 11)
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