15.14 : RNA-seq

RNA sequencing, or RNA-seq, is a high-throughput sequencing technique that identifies and quantifies RNA sequences in a sample. 

It can be used to analyze total RNA or specific RNA populations such as mRNA, tRNA, rRNA and miRNA. The technique has diverse applications in transcriptome analysis, differential gene expression analysis, and RNA editing.

In the case of the analysis of specific populations, the RNA type of interest needs to be isolated from the other RNAs. mRNAs can be isolated using oligo dT probes that are complementary to the poly A tails present on  mRNA transcripts. MicroRNAs,  which are usually around fifteen to thirty nucleotides long, are isolated by size-based extraction methods.

Alternatively, contaminating RNA, such as ribosomal RNA, can be removed using oligonucleotides complementary to the contaminant and covalently linked to magnetic beads. The hybridized ribosomal RNA is separated from the sample using a magnet, leaving behind the RNA of interest.

The purified RNA is used as a template by the enzyme reverse transcriptase to create cDNA, which is further amplified using PCR to create a library for sequencing.

Sequencing can be carried out through one of several available technologies. In one of the most common, cDNA fragments are ligated with short oligonucleotide sequences known as adaptors, which serve as primer binding sites for PCR amplification. Adaptors  may also have unique sequences, called barcode sequences, that are used to tag and identify each cDNA strand.

The library is then amplified using PCR. Following this it is diluted to a low concentration and denatured to single strands with heat, before immobilization on a sequencing chip consisting of oligonucleotides complementary to the adaptors. 

Once attached, the single stranded DNA is cloned using processes, such as bridge amplification, to form clusters of DNA  with the same sequence. This ensures that the strands from an area on the chip are from a single source and emit a uniform signal during sequencing.

Sequencing can be strand-specific or non-strand-specific. In the case of strand-specific protocols , the complementary strand is washed off and the other is used for sequencing.

Fluorescently labeled nucleotides are then added to the strands on the chip to create a new complementary strand. A characteristic fluorescence is emitted on each addition which can be read by a detector. 

Several million clusters of distinct cDNA fragments can be sequenced simultaneously using these methods.The resulting data can then be aligned to a genome of reference and assembled to produce an RNA sequence map for analysis.