WE GO WHERE NO OTHERS CAN
SINGLE MOLECULE miRNA COUNTING BY A PCR FREE METHOD
Hugh Ilyine
CEO Destina Genomics Ltd.
20th February 2020 Manchester Biomarker Conference
DESTINA is a company focused ondelivering high value, high performanceRNA diagnostic assays for diagnosticsand drug development
Small (19 - 25 nt) non-coding RNA molecules that regulate
gene expression at the post-transcriptional level
Specific for pathologies
Altered before clinical symtons appear
Detection from biological fluids
Current methods - Blurred pictures
From analogue to digital
THE RNA BIOMARKER DETECTION DREAM
• No isolation, concentration of targets needed
• Direct detection in all biological fluids, without isolation, concentration of targets –RT-qPCR free
• No refrigeration of samples needed between prep and test, or during transport
• No errors, perfect reproducibility from lab to lab, without concern for inhibitors
• Absolute quantification, not relative quantification, no normalisers needed
• Run in the same machine as protein biomarkers, same sample, same time.
• Fast, cost effective, low technicity required
Taqman PCR and Next Generat ion Sequenc ing
Limitations for microRNA detection:
• Small molecules give errors when
using PCR
• High costs and time-to-results
• Lack of accuracy and lab to lab
reproducibility
PLATFORM AGNOSTIC
Multiple platforms possible• Bead based - colourimetric• Array based – colourimetric, flourescent• Mass based• Magnetic Particle based
High specificity, sensitivity depends on platform used
WHY DESTINA AND AN SMCX-PRO?
• Quantify RNA directly in specimens
• No extraction or amplification required
• Absolute quantification vs relative
• Stabilise RNA at point of collection
• Fast and reliable results
Key – how to dissociate labelled heteroduplex from beads
Hybridization
25ºC
From 10 mL
serum/plasma
DESTINA
Stabiltech
buffer
60 min
1. Bead separation*
2. Washings*
3. DCL Rxn
60 min
1. Bead separation*
2. Washings*
3. Alexa Labelling (20 min)
4. Dissociation (4 min)
30 min
Samples can be stored
and/or shipped at room
temperature (up to 14 days)
* Bead separation and washing steps
done in automatized equipment.
** 96 well-plate formatTarget RNA
Read
RESULTSRobust Cal ibrat ionCur ves
LoD = 1 .5 pM
Sample = 10 uL
N=3
DIL I pat ients serum
14.0 pM to 46 .9 pM
Tr ipl icate per pat ient
Liver-specific microRNA biomarker
Liver disease miRNA signature Expression
Alcoholic liver diseasemiR-122 (acute alcohol, microsteatosis) ↑
miR-122, miR-155 (chronic alcohol, macrosteatosis) ↑
Hepatitis CmiR-122, miR-34a, miR-155, miR-125b, miR-146a, miR-21 ↑
Hepatitis B miR-192, miR-122 ↑
Hepatocellular carcinoma
miR-21 miR-16, miR-199a, miR-122, miR-223, miR-885-5p ↑
Drug overdose miR-122, miR-192 ↑
Degradation of miR-122 is blockedby using DESTINA StabiltechTM
buffer
Stabilises samples at collectionpoints, during transport andsample holding and handlingwithout any refrigeration needed
Multiple miR-122 isomiRs in DILI are less presentin serum from healthy patients
The canonical form of miR-122 was not the mostabundant in both healthy and DILI patients
Sample degradation over time produces moreisomiRs
microRNA degradation was substantiallyincreased if DILI occurs
NGS
A) SYBR Green PCRB) Taqman PCRC) DCL assayD) Virtual patient analysis
DESTINA Genomics Ltd. DESTINA Genómica S.L7-11 Melville St, Edinburgh EH3 7PEUnited Kingdom
+44 (0) 162 082 2611
Avenida de la Innovación 1, 18016 GranadaSpain
+374 660 159 779