微阵列技术和转录组测序技术在肺腺癌病人基因表达改变中的应用

1、 The Comparative Microarray and RNA-seq Analysis of Gene Expression Changes in 10 Patients of Lung Adenocarcinoma and Its Matched Adjacent Noncancer TissuesAbstractDNA microarrays, also known as oligonucleotide array, is a product of the gradual implementation of the Human Genome Project (HGP) and t

2、he rapid development and application of molecular biology, which is inspired by the widely used of computer chip. Itwhich connected financial microelectronics, life sciences, computer science and photoelectrochemical together, developed on the basis of the original nucleic acid hybridization as a ne

3、w technologyis one of a bio-chip. The principle of this technique is integrated known gene probe sequence on a solid surface, a large number of the nucleic acid sequences which are tested in biological cells or tissues hybridized with the probe array hybridized, by detecting the position of a hybrid

4、ization probe to achieve genetic information rapidly.The object of RNA-seq is studying the sum of all RNA transcriptome sequencing come from a specific cell in a functional state that can be transcribed out, including mRNA and non-coding RNA. RNA-seq is the foundation and starting point of the funct

5、ion and structure of genes, through a new generation of high-throughput sequencing, to obtain the species-specific tissues or organs in a state of almost all transcripts sequence information fully and quickly, which has been widely used in basic research, clinical diagnosis and drug development and

6、other fields.The purpose of such experimental design is to suppose that we aim to perform a comparative microarray and RNA-seq analysis of gene expression changes in 10 patients of lung adenocarcinoma and its matched adjacent noncancer tissues. Please design a study on this comparative analysis.Key

7、words：Microarray、 RNA-seq、 lung adenocarcinoma、 gene expression changes.MICROARRAYBackgroundAs the advent of microarray technology since 1995, it is immediately applied to cancer research, and has made many important advances, also has brought hope and possibilities for revealing the occurrence and

8、development of the tumor ultimately. Microarray has a very wide range of applications, such as the detection of gene expression, DNA sequencing, finding a new gene, the detection of mutants and polymorphism, drug screening, disease diagnosis, and gene libraries, and thus it has a great potential and

9、 value about the application of cancer research, especially its high-speed productivity and wealth of information, it will play an important role in determining the tumor risk factors, early diagnosis and timely treatment and prognosis. The application of cDNA microarrays can find specific gene in d

10、ifferent tumor and different period of tine quickly, and provide useful clues for cancer prevention, diagnosis and treatment.Material 1. ChipUsing chips which have 18mm (64 lines), 18mm (64 columns) spotting area and contain 5000 target genes provided by a company. 2. Tissue samplesTen patients with

11、 lung adenocarcinoma from the First Affiliated Hospital of Chongqing Medical University, cancer tissue and normal tissue far from the cancer were taken from surgical specimens, and removed in liquid nitrogen, then moved to -80 refrigerator for RNA extraction. Cancer tissue and normal tissue samples

12、were mixed with related reagents respectively as sample cuvettes, the above RNA samples were prepared for the cancer group and the control group respectively.Method 1. Pre-hybridizationWe put the well-prepared pre-hybridization solution and the chips in a water bath a few minutes for degeneration, d

13、ry out the chips, then add the denatured pre-hybridization solution into to the sampling areas of the chips, then put them into the hybridization-box for 5 to 6 h for hybridizing.2. Labeled the probe We use the direct labeling method of total RNA. Reverse transcriptase was added at RT reaction syste

14、m, then adding the reverse transcriptase buffer, DTT, dNTPs, then, labeling the experimental group with Cy5dC TP in a dark room after reverse transcriptase was added, labeling the control group with Cy3dCTP group in the same way, purifying DNA by DNA purification column, collecting the purified samp

15、le, finally a labeled reagent is added3. HybridWe add the hybridization reagent I in probe tube, mix them thoroughly, dissolve the probe. Then add hybridization reagent II, mix them, take out the chip used as pre-hybridization for reserve, degenerate the probe and chip. We take out the chip and dip

16、them into ethanol, put the probe on the ice after removing it quickly. Then we put the probe on the chip, place them in hybridization compartment, seal them in the hybridized-box overnight.4. WashingWe rinse chip. Preparing two dyeing tanks which are equipped with two kinds of washing agents, immers

17、e the chip in the above two dyeing tank washing in turn, dry and scan the chip.5. Image ProcessingUsing Scan Array 4000 scanner to scan the image, the image processing software is GenePixPro3.0.6. Data ProcessingScreening data which the Ratio (cy5 / cy3) greater than 2 or less than 0.5 as differenti

18、ally expressed genesFlowchartPut the pre-hybridization solution and the chips in a water bath (degeneration) dry out the chips, add the pre-hybridization solution Put them into the hybridization-box，5 to 6h add the reverse transcriptaseAdd the reverse transcriptase buffer, DTT, dNTPs Label the exper

19、imental and control group with Cy5dCTP and Cy3dCTP separately (dark room) purifying DNA(DNA purification column)Add the hybridization reagent I in probe tube I、II, degenerate the probe and chip take out the chip and dip them into ethanolPut the probe on the chip,place them in hybridization compartme

20、nt seal them in the hybridized-box, overnightTwo dyeing tanks (equipped with two kinds of washing agents)Immerse the chip in the above two dyeing tank washing in turndry the chip, scan the chipUse Scan Array 4000 scanner to scan the imageuse GenePixPro 3.0. to process the imageScreening data ResultW

21、e assume there are X gene expression changes in lung adenocarcinoma totally, which include Y raised, Z redused there.RNA-seqBackgroundRNA-seq provides us with the opportunity to detect tumors with full transcript. Such technology makes it possible to allow us to observe the dynamic changes in thousa

22、nds of genes at the transcriptional level, we screen a gene profile of lung adenocarcinoma patients through the high-throughput sequencing technology to get transcriptome gene, and we use RT-PCR to validate, trying to get molecular markers from gene profile which can used for early diagnosis of tumo

23、r molecular changes and prediction of recurrence. Theoretically the changes of mRNA usually earlier than protein levels, and therefore the detection of mRNA expression may be more potential for early detection of tumor recurrence and progression.Specimen CollectionTen cases of Lung adenocarcinoma, n

24、ormal lung tissues far from cancer were taken from surgical specimens, which were taken from the First Affiliated Hospital of Chongqing Medical University, Cutting them into pieces immediately until the size of each side less than 0.5 cm. Placed the pieces into RNA preservation solution, targeted th

25、em, placed them at 4 overnight, save them in refrigerator at -80 to backup.Method1. Real-Time PCR11 RNA extracted We take out samples removed at -80 refrigerator, crush them in liquid nitrogen then add in Trizol so that we extract RNA from tissue, then we detect concentration of the RNA with BIO-RAD

26、 protein assay instrument, determine the concentration and purity ofthe RNA from the absorbance of A260 / A280, and detect RNA integrity by 1% agarose electrophoresis. 12 Reverse transcription into cDNAWe reverse transcription the RNA into cDNA using the reverse transcription kit with DEPC water adj

27、usted to 1 g / L.Reaction System: PrimeScriptBuffer5L Enzyme Mix I2L Oligo dT Prime0.5L Random 6 mers0.5L total added RNA2 LThe product was collected after 15min of reaction at 37, then 5s of reaction at 85, diluted with deionized water, saved in refrigerator at -80 to backup.13 Quantitative PCRWe n

28、eed Quantitative PCR kit and primers to detect the sample, and we detect five genes for each specimen, and each sample repeat tested for three holes.Reaction System: A total reaction system25 L cDNA2 SYBR Premix Ex Taq1 L Upstream primer1 L Downstream primer1 LReaction condition：Pre-denaturation at

29、95 for 5minDenaturation at 94 for 30 sDenaturation at 56 for 30 sExtension 20s Cycle 40 times, drawing melting curve, detecting amplified products with agar gel 2 Statistical analysesUsing SPSS 17.0 statistical software for data analysis, comparing multiple variations among groups, examining the rel

30、ationship between levels of mRNA expression and tumor (initial occur and recurrence) with multiple independent samples of non-parametric test, detecting the differences between differentially expressed molecules validated by RNA expression and transcriptome sequencing, observing expression levels of

31、 candidate genes of 10 cases of Lung adenocarcinoma, normal lung tissues far from cancer, validating data by RT-PCR, processing data with statistical analysis.ResultLogistic regression analysis less than 0.05 was considered as statistically significant resultsReferenses1, SchutzA, Schneidenbach D, Aust G, et al. Differential expression and activity status of MMP1, MMP2 and MMP9 in tumor and stromal cells of squmas cell carcinoma s of the lung J. Tumor Biol, 2002,