Gene expression profiling technology refers to a technology that constructs a non-biased cDNA library of cells or tissues in a certain state, performs large-scale cDNA sequencing, collects cDNA sequence fragments, and qualitatively and quantitatively analyzes the composition of its mRNA population or through high-throughput sequencing of the mRNA produced by a species or a specific cell under a specific functional state to study the gene expression difference, depicting the gene expression type and abundance information of the specific cell or tissue under a specific state.
Gene expression profiling technology has been widely used in the fields of basic scientific research, disease mechanism, biomarkers and drug development. The most widely used at present are gene chips and digital gene expression profiling.
Gene chip refers to the expression level of different genes in the experimental samples obtained by image analysis after the target gene is hybridized with the fluorophore. This technique has great value in diagnosing different cytogenetic subtypes of acute myeloid leukemia (AML), discovering new AML subtypes, and evaluating clinical prognosis. Digital gene expression profiling refers to the ability to generate more than 5M mRNA tags in one sequencing, which can accurately detect low-abundance gene transcripts and simultaneously detect antisense strand transcripts. Compared with the chip technology, there is no cross-hybridization and background noise signal interference. The number of times measured is the expression value of the corresponding gene, which increases the credibility of the data.
During the reverse transcription process, the mRNA of the experimental sample and the reference sample were labeled with different red and green fluorescent groups, and they were mixed to hybridize with the probe sequence on the microarray.
After an appropriate elution step, the chip is scanned with a laser scanner to obtain a fluorescence intensity image corresponding to each fluorescence.
Through the dedicated image analysis software, the red and green fluorescence intensity (Cy5 and Cy3) of each point on the microarray can be obtained, and the ratio (Cy5 / Cy3) is called the expression level of the gene in the experimental sample.
1. Preparation of gene chips
A large number of probes are fixed in a specific arrangement on a carrier such as silica gel, glass sheet, polypropylene or nylon membrane to form a DNA chip. Creative Biomart Biomarker has a variety of chips and a variety of preparation methods to meet your requirements;
2. Preparation of fluorescent labeled probe
Select samples from different states, such as normal tissues and tumor tissues, tissues at different developmental stages, or cells or tissues before and after medication. One of them is called the experimental sample, and the other is correspondingly called the reference sample. The mRNA of the experimental sample and the reference sample to be analyzed are purified, reverse transcribed or amplified grade fluorescently labeled before chip hybridization, and they are mixed to form a gene probe. The most common fluorescent labeling method is to label control samples with Cye3-dUTP (green fluorescence) and laboratory samples with Cye5-dUTP (red fluorescence);
3. Hybridization of labeled probe and chip
According to different experimental purposes, select and optimize the hybridization conditions, hybridize the prepared fluorescent probe with the chip, after a certain time, wash off the unbound probe, and scan to analyze the fluorescent signal;
4. Hybrid chip scanning
The hybridized chip is excited with a laser of a specific wavelength, and the probes on the chip will emit fluorescence at different wavelengths. By detecting the fluorescence intensity of the probe with a confocal laser scanner, two monochromatic images of Cy5 and Cy3 can be obtained. Separate pseudo-color processing of the two images, and then superimpose and call it an image, this is the original data obtained by the experiment-hybrid image.
The fluorescence intensity value of the sample point in the image indirectly gives the relative abundance value of the corresponding probe in the gene chip. If the expression abundance from the test sample is high, the sample point is red; if the expression from the reference cell sample is high in abundance, then the sample point is green; if the abundance is the same, the sample point is yellow; If there is no hybridization reaction between the two, the sample spot remains the background black. In this way, the difference in relative expression levels of genes taken from two different samples can be expressed;
5. Extract the gene expression profile data
Convert the original hybridization image into gene expression profile data
6. Chip result verification method
qPCR, QGP, Northern Blot.