poster summary presentation final
TRANSCRIPT
Development of a combined RT-PCR: NGS assay for microRNA
biomarkers
Centre for Experimental MedicineQueens University Belfast
http://www.qub.ac.uk/research-centres/CEM
Dr William Andrews
MicroRNAs and isomiRs
• miRNAs are small (~22 nt) RNAs• Formed from stemloop precursors • Involved in gene regulation
• Formed by RNAse cleavage of dsRNA
miR-146a
5p
miR-146a
5' 3'
• However, cleavage is not uniform
MicroRNAs as biomarkers
In liver disease
Nature Reviews Gastroenterology and Hepatology 10, 542-552 (September 2013)
Cardiovascular Research (2012) 93, 545–554
In heart disease
So how do we do it?
Oligo-dT linker is annealed to the poly-A tail of the microRNA,incorporating unique molecular counter
Reverse Transcription: cDNA incorporating the reverse primermiRNA-specificity
AAAAAAAAA
TTTTTTTTT
microRNA is polyadenylatedAAAAAAAA
A
microRNA
TTTTTTTTT
PCR amplificationProducts carrying sequencing adaptors
NGS template preparation
5'
3'
5'
3'
5' 3
'
5'
TTTTTTTTT
MC
Detection of isomiRs
% IsomiR from RNA-Seq (Illumina)
% IsomiR from
IzoSeq (Ion
Torrent PGM)
Compatible with Ion Torrent or Illumina
High coverage for miRNAs of interest
Ion PGM sequence Reads miRBase sequence ReadsUAGCUUAUCAGACUGAUGUUG 6064 UAGCUUAUCAGACUGAUGUUGAC 641UAGCUUAUCAGACUGAUGUG 3011 UAGCUUAUCAGACUGAUGUUGA 577UAGCUUAUCAGACUGAUGUAG 2647 UAGCUUAUCAGACUGAUGUUGACUG 124UAGCUUAUCAGACUGUGUAG 2399 UAGCUUAUCAGACUGAUG 123CAGAAGCUUAUCAGACUGAUGUUG 1379 UAGCUUAUCAGACUGAUGUUGACUGU 104UAGCUUAUCAGACUGAUGUUGA 1025 UAGCUUAUCAGACUGAUGU 89UAGCUUAUCAGACUGAUGUGA 638 UAGCUUAUCAGACUGAUGUUG 62UAGCUUAUCAGCUGUGUAG 574 UAGCUUAUCAGACUG 45UAGCUUAUCAGACUGAUGUUGAC 479 UAGCUUAUCAGACUGAUGUU 44UAGCUUAUCAGACUGAUGUAGA 449 UAGCUUAUCAGACUGAUGUUGACU 21
Sequences obtained from IzoSeq sequencing of circulating endothelial progenitor cell (left), alongside miRBase miR-21 reads
(right) for comparison. Sample sequenced has a different isomiR complement to miRBase.
Ion PGM sequence Reads miRBase sequence ReadsUAGCUUAUCAGACUGAUGUUG 6064 UAGCUUAUCAGACUGAUGUUGAC 641UAGCUUAUCAGACUGAUGUG 3011 UAGCUUAUCAGACUGAUGUUGA 577UAGCUUAUCAGACUGAUGUAG 2647 UAGCUUAUCAGACUGAUGUUGACUG 124UAGCUUAUCAGACUGUGUAG 2399 UAGCUUAUCAGACUGAUG 123CAGAAGCUUAUCAGACUGAUGUUG 1379 UAGCUUAUCAGACUGAUGUUGACUGU 104UAGCUUAUCAGACUGAUGUUGA 1025 UAGCUUAUCAGACUGAUGU 89UAGCUUAUCAGACUGAUGUGA 638 UAGCUUAUCAGACUGAUGUUG 62UAGCUUAUCAGCUGUGUAG 574 UAGCUUAUCAGACUG 45UAGCUUAUCAGACUGAUGUUGAC 479 UAGCUUAUCAGACUGAUGUU 44UAGCUUAUCAGACUGAUGUAGA 449 UAGCUUAUCAGACUGAUGUUGACU 21
• Current status: Working Prototype• Academic collaborations
– Exploit increased data content and therefor biomarker potential provided by isoMiR analysis
– Analyse samples for microRNAs of interest• Commercial partners to further develop the
assay
• Speak to us throughout the conference– Poster Number INSERT HERE
Final words
HAPPY SAINT PATRICK’S DAY!