Expression profiling gene chip experiments

Summary

Expression profiling gene chips can be applied to (1) disease diagnosis; (2) new drug development; and (3) environmental protection.

Operation method

Expression profiling gene chip technology

Principle

An array of microdot arrays consisting of ten million nucleic acid molecules immobilized in predetermined positions in a small area on a solid phase carrier. Under certain conditions, the nucleic acid molecules on the carrier can hybridize with nucleic acid fragments from the sample with complementary sequences. If the nucleic acid fragments in the sample are labeled, the hybridization signal can be detected on a dedicated chip reader. Flowchart of basic gene chip technology

Materials and Instruments

Tissue or cell samples
TRIzol Isopropyl Alcohol Ethanol Chloroform dNTPs Hybridization Kit
Electric Glass Homogenizer Electronic Balance Low Temperature High Speed Centrifuge Low Temperature High Speed Benchtop Centrifuge Ultra-clean Bench Ice Maker Electrothermal Thermostatic Bath Electrophoresis Tank Electrophoresis Instrument Microwave Oven Gel Imager Benchtop Centrifuge Nucleic Acid Quantitative Analyzer Pipette Gun Adjustable Electric Furnace Vortex Mixer Hybridization Chamber Hybridization Chamber S-200 Purification Columns Vacuum Concentrator Coverslips Chip Scanner

Move

One, Total RNA extraction
1. Remove the sample bag from the ultra-low temperature preserved samples, weigh them on an electronic balance, transfer them to a milling bowl pre-cooled with liquid nitrogen, and grind the tissues with a pestle and mortar, during which liquid nitrogen is continuously added until they are ground into a powder.
2. Transfer the powdered sample to a homogenizing tube to which the appropriate amount of TRIzol reagent has been added, place the homogenizing tube in an ice bath, and homogenize the sample on a tissue homogenizer. Homogenize until the homogenate is non-stick and free of particles.
3. Transfer the homogenate to a 15 mL centrifuge tube and centrifuge at 12,000 g for 10 min at 4℃.
4. Carefully transfer the supernatant to a new 15 mL centrifuge tube and allow to stand at 15-30°C for 5 min.
5. Add chloroform to the homogenate, cap the tube tightly, shake the tube vigorously, and allow to stand at 15-30°C for 3 min.
6. Centrifuge at 12,000 g for 15 min at 4°C.
7. Carefully remove the tube from the centrifuge and aspirate the supernatant into another 15 mL tube.
8. Add isopropanol to the supernatant, gently invert the tube to mix the liquid well, and allow to stand at 15-30°C for 10 min.
9. Centrifuge at 12,000 g for 10 min at 4°C.
10. Discard the supernatant, slowly add 5 mL of 75% ethanol along the wall of the tube, gently invert the tube to wash the wall, and carefully discard the ethanol.
11. Add another 10 mL of 75% ethanol and vortex briefly on a vortexer; centrifuge at 4°C, 8 000 g for 10 min.
12. Carefully discard the supernatant, centrifuge briefly, aspirate all supernatant with a pipette gun, and dry the precipitate for 5 min in an ultra-clean bench.
13. Add RNase-free Milli-Q water to completely dissolve the RNA precipitate and store at -80°C.
Probe labeling and hybridization
1. Pre-hybridization
(1) Prepare the pre-hybridization solution: add Hybridization Reagent 1 into the Eppenderf tube, shake and mix well, then add Hybridization Reagent 2 and mix well.
(2) Put the prepared prehybridization solution into 95℃ water bath for 2 min, put the slides to be prehybridized into 95℃ water bath for 30 s, put the slides into anhydrous ethanol for 30 s after removing them, and dry them.
(3) Add the denatured prehybridization solution to the spotting area of the slide, cover the coverslip, and place the slide into the hybridization chamber at 42℃ for 5-6 h. The slide was then placed into the hybridization chamber for 5-6 h. The slide was then placed into a water bath at 95℃ for 2 min.
2. Labeling probes (the following in an ice bath)
(1) Add the following reagents sequentially into a sterilized 1.5 mL Eppendorf tube (final volume of the reaction is 50 uL, and the following reagents are RNase-free): _ddH2O 23 uL Reverse transcription primer 5 uL Total RNA 50~100ug Vibrate and mix well, place in a water bath at 70°C for 10 min. remove and quickly place on ice.
(2) Add the following reagents separately:Reverse transcriptase buffer 10uL DTT 5uL dNTPs 4uL
(3) Add the following reagents to the darkroom:Reverse transcriptase 2 uL Cy5-dCTP or Cy3-dCTP 3 uL
(4) Mix the sample by flicking the wall of the tube with a finger and hand-bath for 2 min. place the Eppendorf tube in a water bath at 42°C for 2 h. The sample was then incubated in the dark chamber for 2 h.
(5) Sequentially add 4 uL of Labeling Reagent I to the Eppendorf tube, and add 4 uL of Labeling Reagent II to the Eppendorf tube after a water bath at 65°C for 10 min. mix well, and combine the control and experimental groups. Protect from light and vacuum dry to about 50 uL.
(6) Purify DNA using a DNA purification column (or ethanol precipitation).
(7) Suspend the internally dissolved resin by shaking the column vigorously on a vortex mixer. Loosen the small cap at the top of the column a quarter turn and break the sealing head at the lower end of the column.
(8) Place the column in a 1.5 mL Eppendorf tube, centrifuge at 3 000 rpm for 1 min Place the column in another new 1.5 mL Eppendorf tube, remove the cap at the top, and slowly add the sample to the middle of the upper surface of the resin, taking care not to stir the column. Centrifugation at 3,000 rpm for 2 min resulted in an effluent of purified sample that was collected in a support Eppendorf tube.
(9) Add 8 uL of Labeling Reagent III and vacuum dry.
3. Hybridization
(1) Add 6.5 uL of Hybridization Reagent I to the dried probe tube and mix well to dissolve the probe. Then add 6.5 uL of Hybridization Reagent II and mix well.
(2) Remove the pre-hybridized slides and wash away the coverslips with _ddH2O.
(3) Place the probe in a 95℃ water bath to denature for 2 min; place the slide in a 95℃ water bath to denature for 30 s. Remove the slide and immerse it in anhydrous ethanol for 30 s. After the probe is removed, place it on ice quickly.
(4) Place the probe on the chip, cover it with a coverslip, place it in the hybridization chamber, seal it with Parafilm, and put it into the hybridization chamber at 42℃ for overnight hybridization (16～18 h).
4、Wash the chip
(1) Rinse the slide with 0.5% washing solution1 and remove the coverslip.
(2) Prepare two staining cylinders with 0.5% Wash Reagent 1 + 2% Wash Reagent 2 and 5% Wash Reagent 3, and place them in a 60°C water bath.
(3) Immerse the slides into the above two staining vats for 10 min.
(4) Rinse the slide with 0.5% Wash Reagent 1, air dry and scan.

Common Problems

I. Gene microarray technology steps

Gene microarray technology consists of four main steps: microarray preparation, sample preparation, hybridization reaction and signal detection and result analysis.

1. Chip preparation

At present, the preparation of microarrays is mainly based on glass or silicon as the carrier, and oligonucleotide fragments or cDNAs as probes are sequentially arranged on the carrier using in situ synthesis and micro-matrix methods. The preparation of the chip requires the use of robotics in addition to micromachining processes. So that the probes can be placed quickly and accurately to the specified positions on the chip.

2. Sample preparation

Biological samples are often complex mixtures of biomolecules, and except for a few special samples, they generally cannot react directly with the chip, and sometimes the amount of the sample is very small. Therefore, the samples must be extracted and amplified to obtain the proteins or DNA or RNA in them, and then labeled with fluorescence to improve the sensitivity of detection and the safety of users.

3. Hybridization reaction

Hybridization reaction is a process in which the reaction between the fluorescently labeled sample and the probe on the chip produces a series of information. Choosing the right reaction conditions can make the reaction between biomolecules in the best condition and reduce the mismatch rate between biomolecules.

4. Signal detection and result analysis

After the hybridization reaction, the fluorescence position, fluorescence intensity of each reaction point on the chip can be analyzed by the chip scanner and related software, and the fluorescence will be converted into data, that is, we can obtain the relevant biological information.

Types and characteristics of gene chips

At present, gene chips are mainly composed of oligonucleotide chips and cDNA chips. The basic principles and characteristics of these two types of chips are introduced below:

1. Oligonucleotides Chip (Oligonucleotides Chip)

Concept: It refers to the oligonucleotide microarrays made on the solid-phase carrier. The preparation method is based on direct in situ synthesis on the substrate, or sometimes can be pre-synthesized, and then fixed on the substrate in accordance with the preparation of cDNA chip. In situ synthesis is currently the most successful method for manufacturing high-density oligonucleotide microarrays, and there are several different processes, the most famous of which is the patented technology of Affymetrix (http://www.affymetrix.com). -Light-directed chemical synthesis process. The product is called GeneChip.

The main process of light-directed chemical synthesis disclosed by Affymetrix is as follows: First, the length and sequence of the oligonucleotide probe are determined according to the purpose of hybridization. Then the computer designs all the photomasks (Masks) used in the synthesis of oligonucleotides. Finally, the probe synthesis is done. The advantage of the photoconductive in situ synthesis technique is that an extremely large number of probe arrays can be synthesized with very few steps, and the density of probe arrays can be as high as one million per square centimeter. The main disadvantages of this method are: first, a series of masks need to be pre-designed and fabricated, which is costly; second, the yield of each step is low. Therefore, the length of the synthesized probe is limited.

In addition, the method of original value synthesis is also used by Incyte Phamaceuticals5 (http//www.incyte.com) and Rosetta Biosystem Inc, etc. based on the principle of ink-jet printing IN situ synthesis with reagents delivered by ink-jet printer devices). The ink-jet printer device is similar to a regular color ink-jet printer, replacing the color ink in the cartridge with a four-base liquid, and spraying specific types of reagents onto a predetermined area through a computer-controlled printer. The process of rinsing, deprotection, and coupling is the same as the traditional DNA solid-phase primary synthesis technique. The spray-printing method can synthesize oligonucleotide chains of 40-50 nt in length, with yields up to 99% at each step, and yields of synthesized 30 nt oligonucleotides up to 70% or more. Canon Japan utilizes its original bubble inkjet technology to produce small probe dots of nearly 100 microns on a substrate using only 24 pl of solution. Nearly 20,000 probes can be arranged per square centimeter, overcoming the drawbacks of inkjet printing technology in preparing probe arrays with low density.

Hybridization and detection analysis of oligonucleotide microarrays: Sample processing and hybridization detection methods are consistent with cDNA microarrays. Since oligonucleotide arrays mostly need to distinguish single base mutations. Therefore, strict control of the salt ion concentration of hybridization solution, hybridization temperature and rinsing time is the key to the success or failure of hybridization experiments.

2. cDNA Chip

Concept: Thousands of cDNA molecules fixed on solid-phase carriers such as glass slice, silicon slice, polypropylene membrane, nitrocellulose membrane, nylon membrane, etc. to form a cD4A microarray. The most commonly used solid-phase carriers for the production of cDNA microarrays are microscope slides, which need to undergo surface treatment before use, with the aim of inhibiting the non-specific adsorption of nucleic acid molecules on the surface of the glass slides. Commonly used surface treatment methods include amination, aldolization and polylysine encapsulation.

Preparation of cDNA microarrays: The preparation of cDNA microarrays is mostly done by the Spotting after synthesis method (Spotting after synthesis), or Spotting method for short. Spotting after synthesis method uses special equipment called spotting instrument (Arrayer), at present there are a number of foreign companies (such as Bopdiscovery, Biorobotics, Vartesian Technologies, Genetic Microsystems, Genomicssolutions, etc.) to produce spotting instrument. The main components of the dispenser are made up of The main component of the dispenser is a computerized robot controlled by a computer system. The computerized manipulator uses a pin to dip the cDNA sample from a 96 or 384 well plate and place it on the surface of the slide according to the designed position. The number of needles, the movement time of the manipulator, the cleaning and drying time of the needles, the total number of samples and the number of slides together determine the time required for dispensing; the diameter and shape of the needles, the viscosity of the sample solution, and the surface properties of the solid-phase carriers determine the amount of droplets on the microarray and the diffusion area.

In addition to the spot sampling method, cDNA microarrays can also be prepared by electronic addressing. This technique was first used by Nanogen (http://www.nanogen.com) in the U.S. They electrically activated the holding localization points on the blank slice, so that the surface of the corresponding activation point is charged and becomes a microelectrode, which is able to adsorb cDNA molecules. The slice with microelectrodes is co-incubated with the sample solution, and the cDNA molecules in the solution are adsorbed on the microelectrodes and chemically bonded to the surface of the slice and thus immobilized. The advantage of the chip prepared by this process is that the electrosorption of the microelectrode can improve the hybridization efficiency with the target nucleic acid. The disadvantage is that the preparation is complicated. Higher cost. This chip with microelectrodes is also called active chip. Many companies sell commercialized cDNA microarrays that can be customized from the company as needed. One of the notable cDNA microarray providers is Incyte USA, whose product name is GEMTM microarrays, which can contain up to 10,000 points per slice, with a detection limit of 2 pg of mRNA in the sample, and a 2-fold detection limit of differential gene expression in samples from two sources.

3. How to use cDNA microarrays - sample preparation and hybridization

Sample preparation includes isolation and labeling, and some samples are also amplified by nucleic acid amplification. The general process of sample preparation is: extraction of mRNA in the sample to be examined, reverse transcription into cDNA, while labeled with fluorescence (fluorescent labeling is the most commonly used method. The advantage is that it is non-radioactive and has a variety of colors available; the researcher can choose other labeling methods according to the need, such as isotope labeling, chemiluminescence or enzyme labeling; if the purpose is to study the differential expression of genes in two sources of tissue cells, the mRNA of the two tissues is extracted separately, reverse transcribed into cDNA, and labeled with fluorescent light of two different colors (e.g., Cy3 and Cy5), and then mixed in equal amounts with the chip for heterogeneity. Equal amounts were mixed and hybridization reactions were performed with the microarrays.

The hybridization reaction can be carried out in a dedicated hybridization station or hybridization chamber. The hybridization station can accommodate multiple chips, which is conducive to the automation of the hybridization process and the standardization of hybridization conditions. Individual reactions can be performed in a hybridization chamber, and the laboratory led by Professor Patrick O. Brown at Stanford University provides detailed instructions for making hybridization chambers on the Internet, along with a complete laboratory manual for cDNA microarray equipment, sample handling and hybridization, and downloads of relevant software at http://cmgm.stanford.edu/ pbrowri/index.html.

4. Hybridization signal detection and analysis

Usually the detection of hybridization signals on the chip requires a highly sensitive detection system - Reader, the imaging principle of the Reader is divided into two kinds: laser confocal scanning and CCD imaging. The former has higher resolution and sensitivity, but slower and more expensive scanning speed. The latter holds the opposite. The hybridization information of tens of thousands of points generated by a standard cDNA microarray hybridization experiment requires the support of bioinformatics tools. A variety of applications are available for reading and analyzing hybridization signals, as well as for connecting to public databases on the Web for data analysis. Software for image analysis can be downloaded from the NHGRI's website, and software packages can be found that work online with databases such as Genbank, Unigene, and others.

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