BCH3031 Lecture Notes - Lecture 8: Rna Biology, Non-Coding Rna, Reverse Transcriptase
25 May 2018
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Lecture 8 – Technologies to Understand Transcription and RNA Biology
Concept of Transcriptome
Cellular content of coding and noncoding RNA
• Heavy band/light band in gel
• Vast majority is structural
o rRNA
o tRNA
o snRNA/spliceosomal RNA
• mRNA makes up <10% of RNA
• Some technologies require removal of these
o Poly(A) selection
o Ribosome depletion
o Hybridization based selection
o RNA-immunoprecipitation
Three eukaryotic RNA Polymerases
• Polymerase unwinds DNA to make a copy of RNA
• Pol I
o Makes (precursors) rRNA → 75% of all content of RNA
• Pol II
o Makes primary transcripts, mRNA, miRNA and regulatory ncRNA
(make up less than 10% of RNA)
• Pol III
o Makes small structural RNA, tRNA, snRNA → 15th of RNA
Context specific collection and analysis of RNA
Reverse transcription (cDNA)
• All modern RNA biology involves cDNA synthesis and PCR
o qPCR
o Cloning
o RNA-seq
• Reverse transcription requires
o A Primer (oligo-dT or random primer)
o dNTPs and reverse transcriptase
• Transcriptome analysis by microarray
o Many types: 2 colour, 1 colour
o Probes can be custom designed
▪ Tiled across genome
▪ Tiled across exome (for human)
▪ Detection is hybridization based
o Arrays have whole expressed Transcriptome printed on them
• NGS technologies sometimes ligate adapter sequences directly onto RNA (e.g.
miRNA cloning)
o Transcriptome analysis by NGS
o Library preparation (not in fixed order)
▪ Fragmentation
▪ cDNA synthesis
▪ Adapter ligation or PCR mediated addition of adapters
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