Application of CDK14 gene and expression product thereof
A CDK14 and expression product technology, applied in the field of gene application, can solve the problems of unsatisfactory clinical curative effect of biological therapy, and achieve the effect of accurate diagnosis of liver cancer
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Embodiment 1
[0061] Example 1 RT-PCR experiment detects the expression of CDK14 gene in liver cancer tissue
[0062] RT-PCR refers to a method that combines a reverse transcription (Reverse Transcription; RT) reaction and a PCR (Polymerase Chain Reaction) reaction. RT-PCR combines RNA-templated cDNA synthesis with PCR to provide a fast and sensitive method for analyzing gene expression. RT-PCR is used to detect or quantify expression information. RT-PCR is more sensitive and easier to operate than other RNA analysis techniques including Northern blotting, RNase protection analysis, in situ hybridization and S1 nuclease analysis. The template for RT-PCR can be total RNA or poly(A) selective RNA. The reverse transcription reaction can be initiated with a reverse transcriptase, random primers, oligo(dT), or gene-specific primers. RT-PCR can be performed in a one-step or two-step format. In two-step RT-PCR, each step is performed under optimal conditions. Synthesis of cDNA was first perfo...
Embodiment 2
[0084] Example 2 in situ hybridization
[0085] The CDK14 gene probe was used to carry out in situ hybridization with tumor slices, the stronger the positive hybridization signal, the higher the possibility of suffering from liver cancer. The experimental steps are as follows: the liver tumor tissue is taken, embedded in OCT, quick-frozen in liquid nitrogen, and sectioned at constant temperature, and the frozen section is further used for in situ hybridization. The implementation steps are as follows:
[0086] (1) Frozen section and pretreatment before hybridization
[0087] 1). Take the sample out from -80°C, embed it with OCT, and equilibrate at -23°C (microtome chamber temperature) for at least 30 minutes. Fix the embedded sample on the sample head, cut 10 μm thick serial tissue sections, and spread them on the glass slides coated with poly-lysine (1 mg / ml) (the slides were pre-dried at 180°C for 6 hours) and stored in a -70°C freezer for later use.
[0088] 2). After t...
Embodiment 3
[0112] Example 3 Sequencing the CDK14 Gene Open Reading Frame Sequence in Liver Cancer Patient Tissues
[0113] 1. Extraction of liver cancer tissue DNA
[0114] Use QIAGEN’s DNA extraction kit (QIAamp DNA Mini Kit, Cat.no.: 51306) for relevant extraction operations, see the product manual for details, and the operation steps are briefly described as follows:
[0115] (1) Place the tissue in a mortar, add an appropriate amount of liquid nitrogen to fully grind it into powder, transfer the tissue powder to a 1.5ml centrifuge tube (about 25 mg), add 100 μl lysate and 10 μL RNaseA / protease to the centrifuge tube K stock solution, shake and mix quickly and fully, place in a warm bath at 55°C for 15 minutes, and invert the centrifuge tube several times during this period.
[0116] (2) Add 10 μL of 3M NaAc (pH4.8), and then add 250 μL of DNA Binding Solution, shake and mix thoroughly to make the solution an aqueous solution, and then transfer it to a centrifugal adsorption column. A...
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