Application of DIAPH3 gene and expression product thereof
A technology of gene expression and medicine, application in medicine, application fields of DIAPH3 gene and its expression products
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Examples
Embodiment 1
[0069] Example 1: RT-PCR detection of the expression of DIAPH3 gene in liver cancer tissue
[0070] 1. Tissue separation: The experimental tissue was derived from surgical patients with primary liver cancer. Once the surgically resected liver was isolated from the body, the lesion and surrounding paracancerous tissues 5 cm away were quickly excised and stored in liquid nitrogen (-80°C). The diagnosis of cancer and paracancer is based on pathological diagnosis.
[0071] 2. Extraction of ribonucleic acid (RNA): Total RNA was extracted with Trizol kit (Invitrogen), and the extraction method was referred to the instruction manual of Trizol kit. When RNA is ready to be used, the RNA can be precipitated using the following method: add NaAc to 0.3M, centrifuge at 12,000×g for 5 minutes.
[0072] 3. Synthesis of cDNA:
[0073] (1) Add 4 μL of template RNA (0.5 μg / μL), 2 μL of 20 pmol / μL random primer, 5 μL of deionized water into the ice bath centrifuge tube, mix well, and centrifu...
Embodiment 2
[0085] Embodiment 2: real-time quantitative PCR experiment
[0086] Real-time quantitative PCR was used to detect the expression difference of DIAPH3 gene in liver cancer samples.
[0087] Experimental instrument reagents: Thermal Cycler DiceTM Real Time System TP800, Takara; Premix Ex TaqTM, Takara.
[0088] The designed primers are listed in Table 2, wherein the primers used for DIAPH3 are consistent with the DIAPH3 primers used in semi-quantitative RT-PCR.
[0089] Table 2 Real-time quantitative PCR primers
[0090]
[0091] The real-time PCR reaction system is as follows: total volume 20 μL, SYBR PremixEx Taq 10 μL, upstream and downstream primers (10 μmol / L) 0.4 μL each, cDNA 1 μL, ddH 2 O 8.2 μL, mix the reagents, centrifuge and put them into the PCR instrument for amplification reaction. The PCR conditions are: 94°C for 4min; 94°C for 30s, 60°C for 20s, 40 cycles. Agarose gel electrophoresis was used to detect PCR amplification products, and the results were sh...
Embodiment 3
[0093] Example 3: Detection of the expression of DIAPH3 gene in normal human tissue
[0094] The expression of DIAPH3 gene in normal adult tissues was detected by RT-PCR. According to the method described in Example 1, RNA was extracted from various tissues of normal adults, reverse-transcribed to synthesize cDNA, and β-actin was used as an internal control, and the synthesized cDNA from various tissues of normal adults was used as a template, and the PCR reaction was carried out according to the method described in Example 1. Agarose gel electrophoresis was used to detect PCR amplification products, and the results were shown in image 3 .
[0095] Depend on image 3 The results showed that DIAPH3 was expressed in the embryonic liver, but not in the mature liver.
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