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RNA sequencing

RNA sequencing

RNA sequencing techniques are used to determine the sequence of nucleotide bases, adenine (A), cytosine (C), guanine (G) and uracil (U) in RNA molecules.

Usually RNA is first converted into cDNA (complementary DNA) with a reverse transcriptase enzyme and then a second strand synthesis reaction is performed so that DNA sequencing techniques can be applied to double stranded DNA copies of RNA transcripts. Since this method can lose information from the 5' and 3' ends of the transcript, other methods have been developed that omit the second strand synthesis reaction and ligate adapters to the cDNA for sequencing reactions. Sequencing adapters are consistent sequences that when applied to flank the cDNA fragments produces a cDNA library.

RNA sequencing is replacing gene expression arrays to analyse the spectrum and abundance of transcripts in a given cell or tissue type at a given time. The technique called RNA-Seq, also known as whole transcriptome shotgun sequencing generates cDNA and uses it in next-generation sequencing.

UK-based Oxford Nanopore Technologies devised a system to directly sequence RNA with a device called the MinION, where electrical current is applied across a nanoscale molecular pore and current fluctuations detect the RNA sequence as the RNA molecule snakes through the pore. This RNA sequencing device was used by NASA on the International Space Station because NASA is interested in using it to identify on board microorganisms and to monitor changes in human health or microbiomes and also the possibility of detecting life based on DNA or RNA elsewhere in the universe.

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