Method for isolation of pig lncRNA and identification of specific expression promoter of pig lncRNA
A promoter and specific technology, applied in the field of swine genetic engineering, can solve the problems of reduced tolerance, decreased endometrial regeneration ability, and affected fertility.
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Embodiment 1
[0045] Example 1 Using Invitrogen's Trizol Reagent Reagent to Extract Porcine Related Tissue RNA
[0046] (1) Take an appropriate amount (50-100mg) of pig tissues (heart, liver, spleen, lung, kidney, back muscle, hypothalamus, pituitary gland, ovary, endometrium) into a pre-cooled mortar, and Grind quickly in liquid nitrogen to a fine powder, pour it together with liquid nitrogen into an RNase-free 15ml centrifuge tube, add 1ml Trizol reagent immediately after the liquid nitrogen evaporates, pipette several times, vortex for 1min, and place at room temperature for 5min;
[0047] (2) Add 200 μl chloroform to each tube, cover the sample tube, shake vigorously by hand at room temperature for 15 seconds, and ice-bath for 10 minutes;
[0048] (3) Centrifuge at 12000 rpm for 15 min at 4°C; after centrifugation, the mixture is divided into three phases: the lowermost red phenol-chloroform phase, the middle phase and the upper aqueous phase. Most of the RNA remains in the aqueous pha...
Embodiment 2
[0054] Example 2 Expression profile of pig lncRNAXLOC_2017489 in different pig tissues
[0055] (1) Use the reverse transcription kit from TaKara to synthesize cDNA: add 2 μL of 5×gDNAEraser Buffer, 1 μL of gDNAEraser, 1 μg of Total RNA, and RNase Free dH into an RNase-free centrifuge tube 2 0 to 10 μL. React at 42°C for 2 minutes; then add Buffer 4μL, RT Enzyme Mix 1μL, RT Primer Mix 1μL, add RNase Free dH 2 0 to 20 μL. React at 37°C for 15 minutes, and at 85°C for 5 seconds. The synthesized cDNA was stored at -20°C.
[0056] (2) Using SYBR Green I fluorescent dye (purchased from Invitrogen Company), real-time quantitative PCR was performed in LightCycler480 instrument of Roche Company. Use 2 -△△Ct method for data analysis. The test results showed that pig lncRNA XLOC_2017489 was mainly highly expressed in the endometrium (see figure 1 ).
[0057] The quantitative PCR reaction system and quantitative PCR reaction conditions are shown in Table 1 and Table 2.
[00...
Embodiment 3
[0062] Embodiment 3 Utilizes phenol extraction method to extract pig blood total DNA
[0063] (1) Take a sterilized 50mL centrifuge tube, add 1mL anticoagulant EDTA (0.5mol / L), and add about 20mL of collected pig (large white pig) blood;
[0064] (2) Centrifuge at 3000 rpm for 10 minutes at 4°C, and discard the supernatant serum;
[0065] (3) Add 1.5 times the volume of ddH 2 O, shake gently for 10 minutes to break the red blood cells;
[0066] (4) Centrifuge at 5000 rpm for 10 minutes at 4°C to remove the upper red blood cell plasma;
[0067] (5) Add 20 mL of normal saline for washing, centrifuge at 7000 rpm at 4°C for 10 min, discard the supernatant, and leave the white blood cell pellet;
[0068] (6) Add 1×SET buffer to suspend the cells, add SDS (10%) to a final concentration of 0.5%, then add proteinase K (10 mg / L) to a concentration of 100 μg / mL, and digest overnight at 55°C;
[0069] (7) Add the same volume of saturated phenol, press the tube cap tightly, slowly inv...
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