Non-coding RNA

Major Types of RNA endoribonucleases

Comparative Gene-expression Analysis

Question:Which hybridization technique can be used to detect the amount of polysaccharide?Eastern blottingWestern blottingSouthern blottingNorthern blotting

Agarose gels to resolve large fragments of DNA Polyacrylamide gelsto separate shorter nucleic acids, generally in the range of 1−1000 base pairs, based on the concentration used Gels without a denaturant (e.g., SDS): native gelsSecondary structure affects

Blurry bands Too much DNA (100−250 ng/mm well width) Too much saltBands in the wrong place Heat nucleic acids before running on a native gelRun gel >20 V/cm ( run gel slowly  sharper bands)Gel temp. >30 °CLoading buffer floats away Some salts built up i

Question

Hybridization-based techniques1

Northen blotting

Hybridization-based techniques2

Microarray Platforms

Advantages:Favor small sample sizeHigh throughput: gather data on thousands of genes (genome) in a single experimentQuantitative analysisApplication:Gene expressionGenotyping-polymorphisms (SNP) and copy number variationBinding site identification: ChIP-o

High Throughput of Traditional Experiment

Steps for Microarray Experiment

Overview of the stages

Guidelines

Microarray Platforms

In-House Spotted Arrays

microarray animation

URL for Building Microarrayer

picture of arrayer

microarray arrayer video

HEEBO & MEEBO Genome Sets

Microarray Platforms

Prespotted Array Slides

Question

Phosphoamidite Reaction

Feature (Spot) Morphology

QuestionWhy did the samples need to be co-hybridized onto the same slide in two color platform?To reduce reagent costTo minimize microarray numberTo normalize unequal spot sizeTo simplify experimental protocol

Microarray Platforms

Affymetrix

Probe Set and Probe Pair

CheneChip – Affymetrix

Overview of Target preparation

In Vitro Transcription1

In Vitro Transcription2

QuestionIn DNA structure, coding strand (5’  3’) is also calledWatson strandAnti-sense strandTemplate strandCrick strand

Anti-Sense RNA (Complementary RNA)

Overview of target preparation

QuestionThe purpose of fragmentation during the process of making cRNA is to reduceContaminationCross-hybridizationAmplification copiesFluorescent intensity

Before Hybridization – cRNA Fragmentation1

Microarray Platforms

Maskless Array Synthesis – Nimblegen

Digital Micromirror Device

Maskless Array Synthesis

QuestionWhat similarities and differences are there in manufacturing microarrays between Affymetrix and NimbleGen?

Microarray Platforms

BeadChip--Illumina

Bead Preparation and Array Production

Bead Array: Microwell Fabrication

BeadChips

Bead Design

Decoding Randomly Ordered Bead Arrays

Bead Decoding Example: 16 Bead Types

BeadChip Products

Types of Gene Expression Assays

Types of Gene Expression Assays

DASL Labeling1

DASL Labeling2

Whole Genome DASL Workflow

QuestionWhat is the major differences of Illumina bead arrays as compared to other platforms?

MicroArray Quality Control (MAQC) Project

QuestionWhich value of same samples is smaller?Standard deviation (SD)Standard error of the mean (SEM)

Coefficient of Variation

Repeatability of Expression Signal Within Test Sites

Interplatform Data Comparability

Scatter Plot

Correlation Between Microarray And TaqMan Data

QuestionWhich factors will you consider to choose microarray platform? QualityPriceTurn-over rateAvailabilityAll of them

Decoding Randomly Ordered Bead Arrays