=== [[RNA Sequencing (RNA-seq) === '''RNA Sequencing (RNA-seq)''' is a powerful technology used to analyze the transcriptome of cells, tissues, or entire organisms. The transcriptome includes all RNA molecules expressed within a cell at a specific time and under specific conditions. RNA-seq determines the quantity and sequences of RNA molecules, generating gene expression profiles. The process involves the following steps: #'''RNA Extraction''': #*Total RNA is extracted from the cells or tissues of interest. Total RNA typically includes mRNA, tRNA, rRNA, etc., but RNA-seq mainly focuses on mRNA. #'''cDNA Synthesis''': #*The extracted RNA is converted into complementary DNA (cDNA) using reverse transcriptase. cDNA is stable and suitable for sequencing. #'''Library Preparation''': #*Adapter sequences are attached to the cDNA fragments to prepare a sequencing library. Adapter sequences enable the sequencing machine to recognize and read the DNA fragments. #'''Sequencing''': #*The prepared cDNA library is sequenced using next-generation sequencing (NGS) technology. This process generates millions of short DNA sequence reads. #'''Data Analysis''': #*The generated sequence data are aligned to a reference genome. This step quantifies gene expression levels, detects novel transcripts, splicing variants, gene fusions, and more. '''Applications''': *'''Gene Expression Analysis''': Compare gene expression levels under different conditions or disease states. *'''Discovery of New Transcripts''': Identify previously unknown RNA molecules. *'''Splicing Variant Analysis''': Analyze different splicing forms and variants. *'''Gene Fusion Detection''': Detect gene fusion events, often associated with cancers. '''Advantages''': *High sensitivity and accuracy: Can detect low-abundance RNA. *Broad applicability: Suitable for various biological questions and sample types. '''Limitations''': *Complexity of data processing: Requires advanced computational biology skills to process and interpret large amounts of data. *Cost: High-quality RNA-seq analysis can still be relatively expensive. RNA-seq is an essential tool in biological and medical research, helping to understand disease mechanisms, discover biomarkers, develop new drugs, and more.]]
