DNA Microarray Protocol DNA Microarray Protocol
Basic DNA Microarray Protocol: Osnovni DNA Microarray Protocol:
The Stages in a Microarray Experiment: Pozornica u Microarray Experiment:
1. Obtain DNA Microarray Chip Nabavite DNA Microarray Chip
Currently, over 50,000 cDNA probes can be spotted onto a 25X75mm slide by robotic printing. Trenutno, više od 50000 cDNA proba može biti uočena na 25X75mm slajd po robotika tisak. It is thus possible to array the entire human genome compliment using this high-density array approach (eg Affymetrix Arrays). To je tako moguće razvrstati cijeli ljudski genom kompliment koji koriste ovaj visoke gustoće polje pristup (npr. Affymetrix Razvrstati).
Thousands of distinct spots of known oligos or cDNAs are printed on matrix platforms located on either silicon, nylon sheets, or glass slides. Tisuće različita mjesta poznatih oligos ili cDNAs se tiskaju na matrica nalazi se na platforme ili silicija, najlona sheets, staklo ili dijapozitiva.
2. Cy3 and Cy5 Labeling of RNA Specimens using (RT-PCR) cDNA synthesis Cy3 i Cy5 Označavanje od uzorke koristeći RNA (RT-PCR) cDNA sinteza
The RNA from 2 or more specimens are compared. The RNA from each specimen is reverse transcribed using RT-PCR to cDNA (complementary cDNA). The RNA iz 2 ili više primjeraka su u usporedbi. RNA iz svaki primjerak je reverzni transcribed pomoću RT-PCR na cDNA (komplementarni cDNA). The cDNA is labeled with two different fluorescent dyes which are colored differently (red and green), with Cy3 and Cy5. The cDNA je etiketom s dvije različite boje koje su svijetleći drugačije boje (crvena i zelena), s Cy3 i Cy5.
3. Hybridization of cDNA Probes to DNA array Hybridization of cDNA probe DNA polje
The cDNA probes are hybridized to the DNA microarray. The DNA array is then washed. The cDNA probe su hybridized na DNA microarray. DNA polje je zatim operu.
4. Detection of Hybridization Signal Detekcija signala hibridizacija
Fluorescent hybridization signals are scanned and detected using laser confocal devices. Fluorescentna hibridizacija signali su skenirane i otkrivena koristeći confocal laser uređaje. Data is the filtered and processed. Data je filtrirane i obrađene.
5. DNA Microarray Data Analysis DNA Microarray Data Analysis
Data analysis is conducted using DNA Microarray bioinformatic tools and/or image processing software. Podaci analiza je provedena korištenjem DNA Microarray bioinformatika alata i / ili softvera za obradu slike.
The signal intesities of the spots are correlated with the concentrations of target mRNA samples. The signal intesities od mjesta su korelirani s koncentracije ciljani uzorci mRNA. Data mining is also conducted using statistical programs and algorithms to determine if the gene of interest is up-regulated, down-regulated, or unchanged. Data mining je provedena pomoću statističkih programa i algoritama kako bi utvrdili je li gena u interesu je regulirano-up, down-regulirano, ili nepromijenjene.
Data is then organized using a database. Data je zatim organizirala koristeći baza podataka. Gene Annotation can be then performed on the data, with GO (gene ontology) analysis, clustering analysis (which groups similar genes and pathways together), and analysis of pathways and networks of genes. Gene Komentar može biti izvedena zatim na podatke, sa DALJE (gene ontologije) analiza, analiza skupina (koja grupa sličnih gena i putevi zajedno), i analiza putevi i mreže gena.
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