Quality quantification with qpcr Two-tailed PCR for ultrasensitive detection of micrornas

Transcription

1 Quality quantification with qpcr Two-tailed PCR for ultrasensitive detection of micrornas

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3 Analytical and preanalytical errors are expensive!

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5 European proficiency ring trials Blood DNA & RNA, Blood/Plasm ccfdna Supported by the EFLM (EFCC) Phase 1 Trials Laboratories used their workflows & tools Phase 2 Trials Laboratories will use SPIDIA s optimized workflows Isolated bioanalyties sent back to SPIDIA s laboratories intensive downstream testing Malentacchi F. et al. (2013). Clin Chim Acta 424: % of European routine laboratories had quality parameters out of range Malentacchi F. et al. (2015). Clin Chim Acta, accepted for publication

6 Changes in mrna levels in blood samples

7 log2(rq)* log2(rq)* Effect of stabilization Up-regulated FOSB mrna level Down-regulated TNFRS mrna level Stabilized RT * EDTA 2-8 C EDTA RT -14 Stabilized RT * EDTA 2-8 C EDTA RT PAX-RT EDTA-4 C EDTA-RT PAX-RT EDTA-4 C EDTA-RT * PAXgene Blood RNA Blood Collection Tube D Blood Collection Tube D Malentacchi F et al. (2014). SPIDIA-RNA: Second External Quality Assessment for the Pre-Analytical Phase of Blood Samples Used for RNA Based Analyses. PLoS ONE 9(11): e Zhan H et al. (2014). Biomarkers for Monitoring Pre-Analytical Quality Variation of mrna in Blood Samples.. PLoS ONE 9(11): e

8 Challenges expression profiling using biomarkers Impact of RNA quality? Impact of inhibition? Impact of genomic DNA background? Impact of inter-run variation?

9 Traditionally RNA integrity is tested by electrophoresis RNA extracted from liver tissue. Left at room temperature and analyzed (Bioanalyzer/Experion/Fragment Analyzer) 0min >120min Works quite well, but reflects ribosomal RNA integrity

10 Enzymatic and physical degradation of RNAs in the oocyte Freeze-thaw, incubation in room temp Heat exposure of RNA Curtesy of Radek Šindelka, Insitute of Biotechnology, BTU, Czech Academy of Sciences, CZ Sidova et al., Effects of post-mortem and physical degradation on RNA integrity and quality Biomolecular Detection and Quantification 2015

11 R Q I Physical/chemical degradation 1 0 L -S E xperion system Differential length amplicons (DAmp) D D A m p X -Y Long (L) Short (S) Target F o r m a lin e x p o s u r e ( m in ) Björkman et al., Differential amplicons (ΔAmp) a new molecular method to assess RNA integrity. Biomolecular Detection and Quantification 2015.

12 R Q I Enzymatic degradation C P P IA - E R R m a r k e r ( P ie c e s ) P P IA - E R R m a rk e r (P o w d e r ) R Q I ( P ie c e s ) R Q I (P o w d e r ) Endogeneous RNase Resistant (ERR) marker D D A m p E R R Stability marker T im e in R T (m in ) Björkman et al., Differential amplicons (ΔAmp) a new molecular method to assess RNA integrity. Biomolecular Detection and Quantification 2015.

13 QC of FFPE samples D A m p X -Y : E R R m a rk e r L a n d S a m p lic o n s Paired samples (RNA): Human FF (Fresh frozen) or FFPE preserved cdna synthesis D D A m p X -Y Y = 0, * X - 2 6,9 1 R 2 = 0, A v r C q a ll G O I D A m p X -Y E R R m a rk e r L a n d S a m p lic o n s qpcr Screening with 40+ Gene of Interest (GOI) and DAmp S/L amplicons for: ERR, 18S and B2M D D A m p X -Y F F P E (L -S ) F F (L -S ) A v r C q a ll G O I

14 Spike in controls for inhibition Alien sequence (artificial gene) identical for RNA/DNA RNA DNA

15 PCR vs. RT inhibition

16 Compensate for gdna background 15% of human genes have pseudo genes Pseudo genes usually lack introns Pseudo genes are often present in multiple copies Measure gdna signal in an RT- control reaction ValidPrime + gdna specific assay (ValidPrime) + Reference gdna GOI GOI Cq RT CqRNA log More accurate and more cost effective than RT(-) controls Cq GOI RT Original data gene 1 gene 2 gene 3 gene 4 ValidPrime sample sample sample sample sample gdna standard Cq GOI RT Cq ValidPrime Sample Cq GOI gdna Cq ValidPrime gdna Laurell et al., Nucleic Acids Research, 2012, 1 10; Drug Discovery World (2011)

17 Breaking up a large study into several plates = 384 reactions 384/96 = 4 plates Requires interplate calibration

18 Interplate calibration: stability over time! Interplate calibrators should be measured with very high accuracy. Should be:: Very stable assays Uncomplicated, purified template at fairly high concentration (20 <Cq < 25) Run in replicates (minimum triplicates) The Interplate calibrator shall be stable over time TATAA IPC:

19 Challenges expression profiling using biomarkers RNA quality and it s impact on generated data AMP and ERR Sample data affected by inhibition? Exogenous controls (RNA/DNA spike) Impact of genomic DNA background? ValidPrime Effects of between run variation? InterPlate calibrator

20 Quality control and quality assessment 6426 citations

21 CEN and ISO Technical Specifications Released guidelines (CEN 2015/16 in process at ISO) blood Cellular RNA blood Genomic DNA blood Circulating cell free DNA FFPE tissue DNA FFPE tissue RNA FFPE tissue Proteins frozen tissue RNA frozen tissue Proteins metabolomics in urine, serum and plasma Guidelines in preparation Venous whole blood CTCs: DNA, RNA, stains & proteins Venous whole blood Exosomes: nucleic acids; ccfrna Urine & other body fluids cfdna Saliva Human DNA Saliva and stool Microbiome DNA Frozen Tissue DNA Fine Needle Aspirates (FNAs) DNA, RNA, proteins Metabolomics of body fluids: International ISO Standard FFPE Tissue in situ stainings incl. IHC

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23 Challenges analyzing mirnas micrornas are short (most nt) and cannot fit two conventional PCR primers There is no common sequence feature to use for the enrichment and amplification. The mature mirna sequence is present also in the pre- and the pri-mirnas mirna isoforms (isomirs) might evade capture, due to terminal heterogeneity

24 Current methods make the microrna longer Extension reduces sensitivity One probe only limits specificity

25 Two-tailed RT-qPCR

26 Design concept

27 Sensitivity and dynamic range Sensitive to detect <10 molecules!

28 Sequence specificity across the entire microrna

29 Benchmarking in biological samples

30 2-tube Multiplexing 8 different RT primers were pooled for multiplex reverse transcribed and subsequent singleplex qpcr Δ Cq (relative to singleplex protocol) Sample mir-122 mir-193a mir-1a mir-21a mir-24 mir-30c Let-7a brain cereb heart kidney liver lung muscle average st.dev tube RT-qPCR multiplexing is also possible using probes

31 2-tube Multiplexing 8 different RT primers were pooled for multiplex reverse transcribed and subsequent singleplex qpcr Δ Cq (relative to singleplex protocol) Courtesy of Ben Ayer, Adam McCoy and Luan Sample mir-122 mir-193a mir-1a mir-21a mir-24 mir-30c Let-7a brain cereb Le, Biosearch/LGC heart kidney liver lung muscle replicates average st.dev tube RT-qPCR multiplexing is also possible using probes

32 Discrimination of isomirs

33 Summary: Two-Tailed RT-PCR for microrna New RT-qPCR method High sensitivity Wide dynamic range Very high specificity Unlimited multiplexing in RT with downstream singleplex qpcr RT-qPCR multiplexing with probes

34 Courses at TATAA 3 or 2 days Hands-on qpcr 3 or 2 days Experimental design and statistical data analysis 2 days NGS- Library construction and QC 2 days Digital PCR 2 days Single cell analysis 1 day Quality control in Molecular Diagnostics. New CEN/ISO guidelines 1 day Genotyping with qpcr 1 day qpcr for mirna analysis 1 day Immuno-qPCR 1 day Sample preparation and quality control 1 day Multiplex qpcr 1 day Quality control of qpcr in molecular diagnostics

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