Biology Reference
In-Depth Information
1. Sample preparation:
- isolation of total RNA
- reverse transcription and amplification
- labeling
2.
Hybridization:
- binding between the targets and probes
- washing
3. Detection:
- chip reading
4.
Data acquisition and analysis:
- collection and summary of raw data
- statistical analysis of the data
Fig. 1.2.
Workflow of a DNA microarray experiment.
hybridization is a central process. The sample that contains the targets to
be investigated is added to the DNA chip to allow their binding to the
probes fixed on the chip, resulting in a characteristic-binding pattern
representing the levels of gene expression of the sample. The sample itself
is labeled prior to the hybridization, and the most-often-used labels are
fluorescence labels that allow detection of the binding event. After the
hybridization, the chip is washed and then fluorescence intensities on
the chip are read and recorded by a scanning or imaging device. Raw data
reflecting the fluorescence intensities are statistically analyzed and often
shown by fold changes as compared to control.
In our laboratory, we performed DNA microarray experiments to study
gene expression profiling in mouse leukemia cells. Here is an example of
the procedures for carrying out the microarray experiments. Briefly, cells
are dissolved in RNAlater (Ambion, Austin, TX, USA) and homogenized in
RLT Buffer (RNeasy Micro Kit; Qiagen, Valencia, CA, USA). Total RNA
is isolated by following the protocol for the RNeasy Micro Kit, and qual-
ity is assessed using a 2100 Bioanalyzer instrument and RNA 6000 Pico
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