A transcriptome is an amount of all RNAs transcribed in the particular tissue or cell inside a certain developmental or functional condition, including mainly mRNA and non-coding RNA (ncRNA). The transcriptome studies explore gene function and structure within the overall level and reveal the molecular mechanism in specific biological processes or disease development. Transcriptomic sequencing (RNA-Seq) describes cDNA sequencing by second-generation high-throughput sequencing technology and obtains a whole and rapid use of virtually all transcripts from the given organ or tissue in the certain condition. The transcriptome study could be the basis and beginning point for the research into gene function and structure. Knowing the transcriptome is important to interpret the important facets of the genome and reveal the molecular composition of tissues and cells, plus it plays a crucial role to know the development of the body and illnesses.
Although there are numerous manufacturers of next-generation sequencing machines, the primary concepts resemble. Basically, high-throughput sequencing may be used to acquire RNA sequence information in samples. The higher sequences are detected, the higher the expression level is. While using Illumina sequencing platform for instance, we briefly introduce how a extracted RNA be considered a sequencing library and understanding stored in difficult disk. The following diagram briefly describes the general flow of second-generation sequencing:
General flow of second-generation sequencing
Transcriptome sequencing necessitates preparation of sequencing libraries with specific sequences and lengths which may be directly examined with the sequencer, so we must combine RNA enrichment, fragmentation, reverse transcription, adapters, PCR amplification, length selection, etc. methods for getting sequences with linker sequences (for bridge PCR amplification), Barcode sequences (for distinguishing different samples), and placed cDNA fragments that needs to be tested.
Following a library is made, libraries of numerous samples might be mixed and sequenced. The Illumia sequencing principle is proven inside the figure below. A simple summary is always to integrate the library to the sequencing nick, also to use PCR to amplify only one sequence into clusters to enhance the signal intensity. Your fluorescence signal of each and every cluster is collected and changed into the attached Bases to get sequencing data.
Figure: The Illumia sequencing principle
Using transcriptome technology in biology:
? Quickly obtain mRNA species in addition to their abundance in microorganisms, helping researchers to discover new mRNA isoforms brought on by alternative splicing or alternative polyadenylation site selection
? Analysis of differential mRNA expression information, using the differential expression of gene function analysis. It can be found in cell differentiation, especially embryonic stem cells and neural stem cell differentiation, body development, signal transduction as well as other biological processes inside the overall technique of modifications in gene expression characteristics.
Using transcriptome technology in medicine:
? Through the onset and progression of cancerous as well as other complex illnesses. The intracellular gene expression patterns can alter significantly, and you’ll find significant results in the prevention and treatment of illnesses.