html5-img
1 / 51

Wed 1

suchin
Download Presentation

Wed 1

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

    2. Agenda Introduction to microarray Motivation & previous techniques Concept of biological pathway Northern blot, RT-PCR and real time RT-PCR Affymetrix microarray experiment cDNA microarray experiment Comparison of the two Codelink, Illumina & Agilent MAQC (Microarray Quality Control) Project

    6. Cell cycle

    19. Notes Only Pyrimidines (C and T) have biotin labeled. This is where the color intensities come from. The fragmentation makes the biotin-labeled cRNA shorter and helps efficiency of hybridization. Sequence info of the target mRNA should be known so the complementary sequence can be prepared on the array.

    35. Other platforms of microarray GE Codelink (out of market now) Illumina Agilent

    39. Mechanisms in microarray Important mechanisms that make microarray work: Reverse transcription: mRNA => cDNA. This is usually also the step to label dyes. (Protein can not be reverse translated to mRNA or to another form. So difficult to label dyes.) Double strand binding of complimentary DNA sequences. (Protein does not enjoy such a good property; there are 20 amino acids without complementary binding)

    41. Previous paper in NAR 2003 Evaluation of gene expression measurements from commercial microarray platforms. Tan et al. Nucleic Acids Research. 2003. 31:5676-5684. Poor consistency made it a concern for precise science and routine clinical use.

    42. Experiment Design 7 microarray platforms; each platform implemented in 3 test sites; 4 pools of RNA each with 5 replicates were performed. (3*4*5=60 arrays for each platform) The 4 pools of RNA are: A. 100%UHRR; B. 100%HBRR; C. 75%UHRR + 25%HBRR; D. 25%UHRR + 75%HBRR. UHRR: Universal Human Reference RNA from Stratagene HBRR: Human Brain Reference RNA from Ambion 3 RT-PCR based alternative gene expression platforms are also tested: TaqMan, StaRT-PCR and QuantiGene Assays.

    43. Experiment Design NCI has only 2 test site. AGL has only 2 samples. Some problematic arrays are removed. AGL is not included in this paper. A total of 386 arrays are analyzed.

    44. Difficulties in comparing multiple platforms Each platform has different probe design Sensitivity and specificity of the probes. (some variability of cross-platform may be due to this annotation problem) Database (NCBI RefSeq) often change, making it difficult to match. Probes may bind to multiple alternative spliced transcripts, which may have different functions and expression patterns.

    46. Match genes across platforms All probes mapped to RefSeq and AceView database. Each platform assayed 15,429-16,990 Entrez genes. 23,971 in 24,157 RefSeq NM accessions assayed in at least on platform. Among them, 15,615 accessions (which correspond to 12,091 Entrez genes) were assayed in all platforms. When multiple probes match to one RefSeq, only the probe closest to the 3’ end is used. Finally each platform has 12,091 probes matching to a common set of 12,091 RefSeq from 12,091 different genes.

More Related