A molecular marker for diagnosis and treatment of pituitary tumors
A technology for pituitary tumors and expression products, applied in the field of biomedicine, can solve problems such as low pituitary function, mechanism and mechanism are unclear, and affect the normal secretory function of the pituitary gland
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Embodiment 1
[0062] Example 1 Screening for gene markers associated with pituitary tumors
[0063] 1. Sample collection
[0064] Six samples of normal pituitary tissue and pituitary tumor tissue were collected. All the above specimens were obtained with the consent of the organizational ethics committee.
[0065] 2. RNA sample preparation (using miRNA kit for operation)
[0066] The tissues obtained above were shredded, put into liquid nitrogen and ground into powder, and RNA was extracted and isolated according to the instructions in the kit. details as follows:
[0067] 1) Isolation of RNA:
[0068] A. Add in tissue homogenate or cells Reagent II 1ml;
[0069] B. Stand at room temperature for 3 minutes, add 0.2ml chloroform and shake vigorously for 15 seconds;
[0070] C. Place on ice for 10 minutes;
[0071] D. Centrifuge at 12000g for 15min at 4°C;
[0072] E. Transfer 80% of the aqueous phase into a new 2ml EP tube, add 1 / 2 the amount of absolute ethanol, and shake;
[00...
Embodiment 2
[0087] Example 2 QPCR sequencing to verify the differential expression of PAEP gene
[0088] 1. Large-sample QPCR verification of PAEP gene differential expression. According to the sample collection method in Example 1, 50 cases of normal pituitary tissues and 50 cases of pituitary tumor tissues were selected.
[0089] 2. RNA extraction The steps are as described in Example 1.
[0090] 3. Reverse transcription:
[0091] 1) Reaction system:
[0092] Reagent volume MgCl 2
2μl 10×RT Buffer 1μl RNase-free water 3.75μl dNTP mix 1μl RNase inhibitor 0.25μl AMV reverse transcriptase 0.5μl Oligo-dT Adapter Primer 0.5μl Experimental sample 1μl
[0093] 2) Reverse transcription reaction conditions
[0094] According to the reverse transcription reaction conditions in RNA PCR Kit (AMV) Ver.3.0.
[0095] 42°C~55°C for 60 minutes, 99°C for 2 minutes, 5°C for 5 minutes.
[0096] 3) Polymerase chain reaction
...
Embodiment 3
[0113] Example 3 Overexpression of PAEP gene
[0114] 1. Cell culture
[0115] Human pituitary tumor cell line GT1.1 was incubated at 37°C and 5% CO in medium 1640 containing 10% fetal bovine serum and 1% P / S. 2 , Cultivated in an incubator with a relative humidity of 90%. Change the medium once every 2-3 days, and use 0.25% EDTA-containing trypsin for routine digestion and passage.
[0116] 2. siRNA design
[0117] siRNA sequence against PAEP gene:
[0118] Negative control siRNA sequence (siRNA-NC):
[0119] The sense strand is 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID NO.7),
[0120] The antisense strand is 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO.8);
[0121] siRNA1-PLD5:
[0122] The sense strand is 5'-AACUUCUUUGGAUUCUCAGUC-3'(SEQ ID NO.9),
[0123] The antisense strand is 5'-CUGAGAAUCCAAAAGAAGUUCA-3' (SEQ ID NO.10);
[0124] siRNA2-PLD5:
[0125] The sense strand is 5'-UUGAUCUUGAACUUCUUUGGA-3' (SEQ ID NO.11),
[0126] The antisense strand is 5'-CAAAGAAGUUCAAGAUCAACU-3' (S...
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