Transcriptome refers to the total sum of all expressed genes in a specific cell under specific functional conditions, which includes all transcript information of a cell. Typically, transcriptome analysis focuses on mRNA. Transcriptome sequencing and analysis can be used to discover low-abundance transcripts, identify polymorphic markers, deeply mine new genes, draw transcript maps, identify gene families, regulate alternative splicing, determine metabolic pathways, and conduct evolutionary analysis, which are particularly important as molecular biology enters the application stage.
Transcriptome sequencing gene chip technology
During the "Human Genome Project," gene chip technology rapidly developed and was widely used as one of the most important research methods of functional genomics. In 1991, the world's first oligonucleotide gene chip was developed based on nucleic acid hybridization. After many years of development, gene chip technology has become relatively mature. The analysis speed has been improved and the required samples and reagents for experiments have been reduced. The experimental technique and subsequent data analysis are quite mature, and a huge public database has been formed. The disadvantage is that the information of the probes on the chip determines the detection range of the gene chip, and this technology is only applicable to detecting known sequences, not exploring the effects of new genes. Additionally, its hybridization technology has low sensitivity, making it difficult to detect low-abundance genes or capture subtle changes in gene expression levels.
Serial analysis of gene expression (SAGE) technology for transcriptome sequencing
The technical process of SAGE technology is to use the anchored enzyme to cut the double-stranded DNA and connect the corresponding adapter. Then, the tag enzyme is used to cut the SAGE tag and amplify it. The adapter sequence is removed using the anchored enzyme to obtain a poly-nucleosome containing the tag dimer, which is then sequenced. SAGE technology is based on Sanger sequencing and has the advantage of quickly obtaining transcript maps.
Massively parallel signature sequencing (MPSS) technology for transcriptome sequencing
The improvement of SAGE technology resulted in MPSS sequencing technology. First, the cDNA is cloned into a vector library with different adapters, and then each cDNA fragment in the vector library is amplified by PCR. Then, the PCR product is converted into a single-stranded library by the joint action of polymerase and dGTP. Finally, the library is hybridized with micro-carriers and sequenced. MPSS technology can detect the expression of all genes in a tissue or cell in a short time and is an effective tool in functional genomics research.
RNA sequencing technology (RNA-Seq) for transcriptome sequencing
RNA-Seq is the most widely used transcriptome sequencing technology developed in recent years, and it has many advantages.
It has high resolution. Transcriptome sequencing technology can accurately distinguish a single base, and issues such as background noise and cross-reactions caused by fluorescent simulation signals can be effectively avoided.
It has high throughput. Transcriptome sequencing technology can obtain hundreds of millions of nucleotide sequences, covering the entire transcriptome.
Iit has high sensitivity. This sequencing technology can even detect rare transcripts with as few as a few copies in the target cells.
It is more convenient to use. This technology can analyze the entire transcriptome of a species without the need to design specific probes before sequencing, directly analyzing the entire transcriptome of the species.
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