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In order to make it easy for everyone to understand quickly, I consulted a lot of materials and presented common genomics schemes and concise experimental steps in the form of words and pictures. In this issue, we shared the marker detection methods of genomics, protein genomics, metabonomics and transcriptomics. Cut the crap and go straight to the dry goods.
1. Marker verification experiment of genomics
1.？ Gene expression
Reverse transcription quantitative PCR(qRT-PCR) is a model to study gene expression in the case of small or precious sample size. The main advantages of this method are wide range, high sensitivity, high accuracy, no PCR post-processing step, avoiding cross-contamination, high yield and multiple detection. Specifically, the quantitative and qualitative analysis of the initial template is realized by detecting the fluorescence signal of each circulating product in the PCR amplification reaction in real time. Fluorescent chemicals are introduced into the real-time fluorescence quantitative PCR reaction. With the progress of PCR reaction, the products of PCR reaction accumulate continuously, and the intensity of fluorescence signal increases in the same proportion. After each cycle, the fluorescence intensity signal was collected, and the change of product quantity was detected through the change of fluorescence intensity, and finally the fluorescence amplification curve was obtained. See reference [1] for experimental ideas.
experimental materials
Fresh and frozen tissues and cells, fresh and frozen blood or plasma, etc.
? Detection methods of gene expression also include PCR[2] and RT-PCR[3].
2.？ Methylation detection
PCR (methylation-specific PCR (MSP)) is a highly sensitive DNA methylation detection technique, which is not limited by restriction enzymes. Usually, two pairs of primers are designed, one pair of MSP primers amplifies the DNA template treated with bisulfite, and the other pair amplifies unmethylated fragments. If the first pair of primers can amplify the fragment, it means that the detection site is methylated; if the second pair of primers can amplify the fragment, it means that the detection site is not methylated. See reference [4] for experimental ideas.
experimental materials
Fresh, frozen and paraffin embedded cells or tissues, etc.
? Other technologies for methylation detection include bisulfite treatment+sequencing [5], restriction endonuclease analysis combined with sodium bisulfate (COBRA)[6] and so on.
2. Verification experiment of protein omics markers.
Western blot is a protein detection technique that transfers the total proteins of cells or tissues separated by electrophoresis from gel to solid support NC membrane or PVDF membrane, and then detects a specific antigen with specific antibody. Its basic principle is to color cells or biological tissues treated by gel electrophoresis with specific antibodies. The experimental method is widely used, which is a common qualitative and quantitative method for protein detection. See reference [7] for experimental ideas.
experimental materials
Fresh, frozen cells or tissues
Enzyme-linked immunosorbent assay (ELISA) is a method for qualitative and quantitative detection of immune response by using the specific binding of antigen and antibody. Basic principle: make antigen or antibody combine with the surface of solid carrier to maintain its immune activity. The antigen or antibody is linked with an enzyme to form an enzyme-labeled antigen or antibody, and the enzyme-labeled antigen or antibody retains its immune activity and enzyme activity. During the determination, the detected sample and the antigen or antibody labeled by enzyme react with the antigen or antibody on the surface of the solid carrier according to different steps. The antigen-antibody complex formed on the solid carrier was separated from other substances by washing, and the amount of enzyme finally bound to the solid carrier was proportional to the amount of detected substances in the sample. After adding the substrate of enzyme reaction, the substrate is catalyzed by enzyme to become colored products, and the amount of products is directly related to the amount of detected substances in the sample, so qualitative or quantitative analysis can be carried out according to the depth of color reaction. See reference [8] for experimental ideas.
experimental materials
Fresh, frozen cells or tissues
Immunohistochemistry (IHC) is also called immunocytochemistry. It applies the principles of immunology and histochemistry to detect the protein level in tissue sections. It uses the labeled specific antibody (or antigen) to detect the distribution of antigen (or antibody) in tissues and cells in situ, detect the corresponding target antigen (or antibody), and make localization, qualitative and quantitative analysis. According to the properties of markers, detection techniques can be divided into immunofluorescence technique, immunoenzyme technique, immunometal technique and radioimmunoassay. Immunofluorescence technology and immunoenzyme technology are widely used in the experiment. See reference [9] for experimental ideas.
experimental materials
Paraffin-embedded tissues, cell specimens and frozen tissue sections
3. Metabonomics Marker Verification Experiment
? Gas chromatography-mass spectrometry (GC-MS): Gas chromatography uses different partition coefficients of different substances in stationary phase and mobile phase to make different compounds flow out of the chromatographic column at different times, thus achieving the purpose of separating compounds. Mass spectrometry uses the motion law of charged particles in magnetic or electric fields to separate and analyze the charged particles according to their mass-to-charge ratio (m/z) to determine the ion mass and intensity distribution. It can give the molecular weight, elemental composition, molecular formula and molecular structure information of compounds, and has the characteristics of qualitative specificity, high sensitivity and rapid detection. See reference [10] for the experimental idea.
experimental materials
Small molecule, volatile, thermally stable and vaporizable compounds
? Liquid chromatography-mass spectrometry (LC-MS): It has the advantages of wide analysis range, strong separation ability, reliable qualitative analysis results, low detection limit, fast analysis time and high degree of automation. It uses liquid chromatography as a separation system and mass spectrometry as a detection system. The sample is separated from the mobile phase in the mass spectrometry part, and the ion fragments are separated by the mass analyzer of mass spectrometry after ionization, and the mass spectrometry is obtained by the detector. See reference [1 1] for the experimental idea.
experimental materials
Non-volatile, polarized, thermally unstable and macromolecules (protein, peptides, polymers, etc. )
4. Verification experiment of transcriptome markers
1.？ Expression verification
QRT PCR (see genomics) and Northern blot.
Northern blot is used to detect and quantify whether the sample contains the transcription product (mRNA) of the gene. The principle is that the RNA sample to be detected is subjected to agarose gel electrophoresis under denaturing conditions, and then the position in the gel is transferred to nitrocellulose membrane or nylon membrane, and then it reacts with RNA probes labeled with isotopes or other markers after fixation. There is little applied literature. See reference [12] for the experimental idea.
experimental materials
Total RNA sample or mRNA sample (RNA is easy to degrade, so it is necessary to pay attention to the preservation method and cycle)
Sensitivity: qRT-PCR & gt；; Northern blot, specificity: Nortern blot & gtQRT-PCR (the above experimental scheme).
2.？ Interaction between RNA and protein
RNA immunoprecipitation (RIP): The RNA binding protein (RBP) of interest is immunoprecipitated with its binding RNA to identify the RNA that binds to transcription. It can be detected by RT-PCR, microarray or sequencing, and it is an antibody-based technique for locating RNA- protein interaction in vivo. See reference [13] for the experimental idea.
experimental materials
cell
RNA- pull-down: RNA is labeled (such as biological probe labeling) and incubated with cytoplasmic protein extract to form RNA- protein complex. Protein was separated by SDS-PAGE, and protein was detected by protein blot or mass spectrometry. Used to find protein binding to target RNA. See reference [14] for the experimental idea.
experimental materials
Cytoplasmic protein
? In addition, it also includes methylated RNA immunoprecipitation (MeRIP)[ 13].
Today's sharing is over. Because there are many ways to verify each group of tags, this issue mainly shares common schemes. I hope you can have a simple understanding of the experimental methods of each group of markers. Based on the follow-up mechanism of each group, it is necessary to add cell proliferation, cell migration, invasion, apoptosis experiments and animal models. Most of them are similar. I believe that you will find the law soon after reading the relevant literature ~
Finally, I wish everyone that every manuscript of SCI will win an award!
refer to
1.？ Liu X, Wang J, Chen M, Liu S, Yu X, Wen F. Identification of gene prognostic markers of lung cancer by combining data from GEO database and reverse transcription quantitative PCR. Onco's goal is there. 20 19; 12:709‐720. Published on 20 19 1 month 2 1 day. doi: 10.2 147/OTT。 S 183944
2.？ Analysis of bacterial distribution in tumors and adjacent normal tissues of 9 major cancer types by TCGA exon sequencing? Journal of computational and structural biotechnology? vol . 18 63 1-64 1。 March 2020, doi:10.1016/j.csbj.2020.03.003.
3.？ HIV- 1 Nef induced lncRNA AK006025 regulates the gene expression of cxcl9/10/1cluster in astrocytes by interacting with CBP/P300. ? Neuroinflammation. 20 18; 15( 1):303. Published on 20 18 Oct 3 1. doi: 10. 1 186/s 12974-0 18- 1343-x
4.？ Xie Kai, Zhang Kai, Kong Jun, etc. Cancer-testis gene PIWIL 1 promotes the proliferation, migration and invasion of lung adenocarcinoma cells. ? Cancer medicine. 20 18; 7( 1): 157‐ 166.doi: 10. 1002/cam 4. 1248
5.？ Abnormal methylation of mutL gene homologue 1 is associated with increased risk of non-small cell lung cancer. ? Clinical laboratory analysis. 20 18; 32(5):e22370。 doi: 10. 1002/jcla . 22370？
6.？ GALNT9, BNC 1 and CCDC8 genes are often epigenetic disorders in breast tumors that metastasize to the brain. ? Clinical epigenetics? Volume 7, 1 57. May 27th. 20 15，doi: 10. 1 186/s 13 148-0 15-0089-x
7.？ Napabucasin, a new STAT3 inhibitor, inhibits the proliferation, invasion and differentiation of glioblastoma cells. ? Clinical cancer research 2019; 38( 1):289. Published on July 5, 20 19. doi: 10. 1 186/s 13046-0 19- 1289-6
8.？ Serum CCL20 combined with IL- 17A is a biomarker for early diagnosis and prognosis of colorectal cancer. ? Medical journal. 20 19; 17( 1):253. Published on August 6th, 20 19. doi: 10. 1 186/s 12967-0 19-2008-y
9.？ The genomic targets related to N6- methyladenosine in breast cancer tissues have changed and are associated with low survival rate. ? J cancer. 20 19; 10(22):5447‐5459. Published on August 29th, 20 19. doi: 10.7 150/JCA . 35053
10.？ Chen Hong, Pan Ding, Yang Zhi, Li Li. Master's degree thesis of China Agricultural University. RAB23？ Show the function of. RAB23？ Gastric adenocarcinoma. ? Ann Transl medicine. 20 19; 7(23):745.doi: 10.2 1037/atm
1 1.？ Pathway analysis based on integrated genomics reveals that CYP cyclooxygenase-related network is the diagnostic target of metastatic triple-negative breast cancer. ? Clinical cancer research 2019; 38( 1): 187. Published on May 9, 20 19. doi: 10. 1 186/s 13046-0 19- 1 187-y
12.？ Wang Xiaolin, Shi Weiping, Shi Huichun, et al. Down-regulation of TRIM65 inhibits the proliferation, migration and invasion of lung cancer cells: the target of lung cancer treatment. ? Oncotarget。 20 16; 7(49):8 1527‐8 1540.doi: 10. 18632/onco target . 13 13 1
13.？ WTAP promotes the progression of hepatocellular carcinoma through m6A-HuR dependent epigenetic silencing of ETS 1. ? Mol cancer. 20 19; 18( 1): 127. Published on August 22nd, 20 19. doi: 10. 1 186/s 12943-0 19- 1053-8
14.？ Lncrna epb41l4a-as1regulates glycolysis and glutamine decomposition by mediating nucleolar translocation of HDAC2. ? EBioMedicine。 20 19; 4 1:200‐2 13.doi: 10. 10 16/j . ebiom . 20 19.0 1.035
15.？ Transcription as a Means of Experimental Verification after high-throughput sequencing (I)