Multiplexed Tandem PCR

Transcription

1 Multiplexed Tandem PCR Operation and Applications Gene expression or SNP profiling from picograms of RNA - Multiplexed Tandem PCR Keith Stanley PhD 1 Keith Stanley Director of Molecular Research Corbett Research Background Diagnostic applications often need to work on small samples, eg a swab or section of a paraffin embedded biopsy. Diagnostic applications typically need multiple genes. Current instrumentation does not provide a turnkey solution - ie skilled staff are required to run and analyse tests. 2

2 Multiplexed Tandem-PCR Multiplex 96 genes Multiplex Step 1 PCR for 15 or 20 cycles Nested PCR gives high specificity 2-step reaction gives molecular sensitivity Dilute Step1 reaction mix into Gene-Disc Nucleic Acid Research 33, e180 (2005) 3 Profiling 36 genes in duplicate from 10 ng of RNA Bioanalyser trace of products A typical MT-PCR reaction produces parallel cycling curves and products that lack primer dimer, allowing quantitation using intercalating dyes. 15 cycles of step 1. 4

3 Semi-automatic analysis in Rotorgene Sample + Quantace mastermix Step 1 template, Rotor-Gene Step 1 Product Rex file Dilute in Mastermix Pipette into Gene-Disc Step 2 template, Rotor-Gene Analysis template, Rotor-Gene Result with automatic calling 5 Gene - Plex robot deck 6

4 MT-PCR diagnostics use Templates for automatic operation Temperature profile for Rotor-gene RT Pre-amp Dilution Quantitation HRM 90 min Sample throughput is approx 15 min per sample for 6 x 12 gene diagnostic 7 MT-PCR analysis reports Autocalling of results using MT-PCR enabled Rotorgene software and analysis template from AusDiagnostics.com 8 4

5 MT-PCR can detect a single molecule Bioanalyser and melt curves show correct product is made, even with a single molecule input. A cloned target cdna was diluted in a log series and analysed by MT-PCR with 20 cycles preamplification genes in duplicate from 10 pg 27 genes correctly detected 20 cycles of step 1 amplification 10

6 Reproducibility GENES Mean SD CV ESR TOP2A CCND PTEN MDM TP VEGF MYC PgR BSG GSTM MKI MELK MAD2L BUB TPD HPRT NAT E2F TGFB TGFB SMAD NFkB BTF Mean ng MCF7 RNA 10 cycles, Raw Ct values Reproducibility of extraction and MT-PCR 10 4 Gene Expression adjacent sections of xenograft extracted with AusDiagnostics FFPE RNA extraction kit and measured by MT-PCR. Gene expression is measured over 5 logs and is constant between sections Section 12

7 Reproducibility of FFPE extraction/mt-pcr 10 Normalised Gene Expression 1 Each gene expression value was normalised to the mean for that gene in all sections. Each data point represents the mean of 30 genes with > 0 expression normalised in this way. Error bars indicate standard deviation Section Cv = 0.3 for all 30 genes 13 Correlation with qpcr 1000 Ratio MCF7/SKBR3 100 R= qpcr MT-PCR 14

8 Quantitation R = and 50 ng input MCF7 RNA with 15 cycles preamplification 15 Features of MT-PCR Nested protocol gives high specificity for target Short target gives independence from degradation Preamplification gives high sensitivity (eg single cell) Works with cheap SYBR green chemistry (research) Confirmation of ID using probes or HRM Works for any primers without customisation Can multiplex at least 96 genes 16

9 High Resolution Melt Analysis Melting curves-normalized by selecting linear regions before and after the melting transition 17 HRM can detect a single base pair change 18

10 MT-PCR as front end to HRM analysis Outer Inner MT-PCR Bioanalyser Gel view Guaranteed clean product in similar concentration Melt modifiers in original sample diluted out HRM performed in defined medium 19 PCR product confirmation by MT-HRM Identification using labelled probes Single bp change may not alter probe hybridisation Identification using MT-HRM 4 primers and comparison with ondisc standard over 30 temperature points 20

11 Drug resistance mutations of influenza virus Sample Gene Sample Type Wild type Mutant Call 1 N1 H274Y Wild type Wild type 1 N2 E119V Wild type Wild type 1 N2-R292K Wild type Wild type 2 N1 H274Y Mutant Mutant 2 N2 E119V Mutant Mutant 2 N2-R292K Mutant Mutant 19 N1 H274Y Sample N2 E119V Sample Mutant 19 N2-R292K Sample N1 H274Y Sample N2 E119V Sample 77 0 Wild type 21 N2-R292K Sample 0 0 Black = standard, red = clinical sample Clinical samples (19 and 21) compared to standards (1 and 2) and N2 E119V spiked HIN1. 21 Analysis of influenza species using HRM and q-pcr Sample Gene Con (%) Yield (%) Call 6 HA-H Present 6 HA-H3 1 Bad Melt 6 HA-H HA-H Pan-INF-A Present 6 Pan-INF-B RSV NONO SPIKE Present HA-H1 HA-H3 HA-H5 HA-H5 2 Pan-INF-A Pan-INF-B RSV NONO SPIKE Name Present / Absent Normalised Expr. HA-H1 Present HA-H3 - HA-H5 - HA-H5 2 - Pan-INF-A Present Pan-INF-B Present 54.2 RSV - NONO - SPIKE Present HA-H1 HA-H3 HA-H5 HA-H5 2 Pan-INF-A Pan-INF-B RSV NONO SPIKE

12 Fungal Triage pan Fungus pan-candida Scedosporium prolificans pan-fusarium C.albicans C.glabrata C.krusei C.paraps C.dublin C.tropicalis C.guiliermondi SPIKE Melt analysis of Fungal Triage products SPIKE C.albicans pan-fungus pan-candida 23 Bacterial resistance genes and ID Pan-Staphylococcus meca nuc gsea (S.epidermidis) VanA (vancomycin) VanB Pan-Streptococcus Ply (pneumolysin) lyta Pan-Pseudomonas Pan-Enterobacteriacea SPIKE 24

13 Summary MT-PCR is an enabling technology for high throughput gene expression profiling with PCR accuracy. MT-PCR is especially suitable when profiles are needed from very small samples of RNA. MT-PCR is an ideal front end for HRM analysis MT-PCR provides a new generation of diagnostics with increased information at similar cost to current devices. Custom MT-PCR assay design and evaluation Preassembled MT-PCR assays 25