WP2 – ddPCR and microRNA C.Divieto, INRIM, Torino, Italy

From DNA to microRNAs DNA that does not make proteins DNA that does make proteins

m rRNAs tRNAs snRNAs ncRNAs Coding RNA NON Coding RNA 22 nucleotides DNA: from 500 thousand to 2.5 million nucleotide pairs Coding RNA ncRNAs 200 nucleotides NON Coding RNA

Rule in normal biological processes: regulation of cell cycle control, neurogenesis, aging Rule in diseases: cancers, heart diseases and neurological diseases. miRNA in AD: more than 100 miRNAs related to AD; Traditional monitoring by invasive CSF collection miRNA circulation in blood: non-invasive collection microRNAs regulate the expression of half of all human genes by binding messenger RNA and preventing their translation into proteins. MicroRNAs are involved in several biological processes: from cell cycle control, to developmental and physiological processes including neurogenesis, aging, stem cell differentiation, hematopoiesis, hypoxia, cardiac and skeletal muscle development, insulin secretion, cholesterol metabolism, immune responses and viral replication. In addition, highly tissue-specific expression and distinct temporal expression patterns during embryogenesis suggest that microRNAs play a key role in the differentiation and maintenance of tissue identity.

Non invasive analysis of microRNAs 2.

From microRNAs to cDNA to be amplified 3. Polymerase Chain Reaction (PCR) DNA amplification 4.

number of DNA molecules Digital Droplet PCR Number of droplets number of DNA molecules at the origin Absolute value  no calibrator needed More PCR reactions (up to 20,000)  improved statistics and accuracy Low limit of detection  high power to discriminate small differences Number of samples in the same amplification  8 to 96 Microfluidic technology  controlled volume of partitioning Droplets are read one by onel, as they pass through a single line channel, by two-color optical detection system in a serial manner. The PCR-positive and PCR-negative droplets are counted to provide absolute quantification of target DNA in digital form.

Results on microRNA by Digital Droplet PCR Limit of detection and specificity of miRNA assay with synthetic miRNA 26a-5p Sample Target DNA copies/uL expected measured Inter - expts CV Intra - expts CV Synthetic miRNA (Exiqon) mixture miR-26a-5p 100 97.7 ± 7.6 7.9 3.7 n=3 expts 50 49.0 ± 3.6 7.4 3.8 n= 3 repeat 10 11.5 ± 0.5 4.4 2.6 5 6.2 ± 1.0 16.9 12.1 Sample Target Dilution factor of extracted RNA DNA copies/uL Expected dilution ratio Measured dilution ratio 1 donor blood miR-26a-5p 50 43.8 ± 0.7 / n=3 expts; 100 24.1 ± 2.3 2.0 1.8 n= 3 repeat 200 9.6 ± 0.6 2.5 400 4.9 ± 0.2 Inter-exp CV ≤ 20.7%; Intra-exp CV ≤ 9.7% Next step: analysis on RNA from ̴45 already recruited patients On-going comparison with immunoassay