Template Quality and Realtime

Transcription

1 Tempate Quaity and Reatime QPCR Infuence of tempate quaity on rea-time QPCR resuts Cathy Cuter Fied Appication Scientist

2 Steps towards successfu Rea-time QPCR experiments 3. RNA/DNA quantification and quaity 1.Experimenta Design 2.Sampe preparation and purification 6.Post-run Anaysis 5.Rea time QPCR AAAA AAAA AAAA Tota RNA cdna 4.Reverse Transcription 2

3 Why Quaity Matters RNA/DNA quaity contro (Quaity of tempate): RNA degrades naturay due to enzymatic or autocataytic mechanisms: Any 5 or 3 biased design might fai or produce miseading resuts Wrong priming strategy in the RT step can ead to miseading resuts Knowing RNA/DNA quaity aows to accommodate the design and set expectations avoiding wrong interpretation of resuts A quantifications rey on comparabe tempate quaity to be meaningfu QPCR assay vaidation (Quaity of resuts): The resoution of SYBR Green metcurves is imited T m depends on dye/tempate ratio SYBR Green is a non-saturating dye with non-uniform distribution aong the doubestrand Verifying the size of PCR products is a recommended vaidation procedure Resoution of sab ges again is imited! 3

4 Sampe Purification Sampe purification greaty infuences QPCR resuts: Buffers from sampe purification can interfere with downstream RT or QPCR (eg. ce cuture medium, etc.) Co-purified sats infuence primer/probe binding characteristics Co-purified inhibitors can ead to faiure of PCR (eg. Pheno, poy-saccharides, heme, ipids, heparin etc.) Timing and way of purification can impact tempate quaity Optimizing sampe purification enhances QPCR efficiency and success 4

5 Sampe Purification RNA 1 human ce contains: 10-5 µg RNA 80-85% rrna (5-, 5,8-, 18-, & 28S) 15-20% ow-moecuar weight abundant RNA 1-5% mrna RNA Isoation not so different from DNA isoation Probem is not the instabiity of RNA, but the stabiity of RNases mrna vs. Tota RNA purification: - Not a mrnas have a poy-a tai - Length of poy-a tai varies Affinity of mrnas to oigo-dt based purification systems varies! mrna purification might introduce a bias 5

6 Sampe Purification Chaenges with FFPE sampes Fixation aters the nuceic acids: Crossinking between protein and nuceic acid fragmentation Purification methods time consuming (severa hours over night) RNA can be highy degraded: Don t use oigo-dt for RT priming Use of very sma ( 100 bp) ampicons Fresh Frozen FFPE Frozen tissue FFPE tissue 6

7 Sampe Purification Chaenges with FFPE sampes Sampe ProtK μg RNA Method 1 1.5h 8.2 Absoutey FFPE 16h 3h 16h 9.2 Competitor 3h 16h 16h Absouty RNA FFPE Pheno Competitor 1.5h/16h Pheno 2 1.5h h 32.2 Competitor NTC 1.5h h 16.3 Absouty RNA FFPE 16h 47.4 Pheno 7

8 WhyDNA Quaitymatters DNA degradation in preserved bioogica tissue, forensic sampes or sampes commony used in pathogen detection can negativey impact assay performance and produce miseading resuts Reduced popuation of DNA with fu ength of ampicon: Underestimation of quantity Competition by abortive ampicons: Loss of sensitivity or inhibition Assay performance and success Sma DNA fragments compete with primers: Unspecific ampification, Aterations of bases: Reduced affinity of primers and probes 8

9 Comparative Quantification Given two sampes: What is the difference in gene expression? Caibrator (Contro) Unknown 1 Unknown 2.. Gene of Interest Reference gene (Normaizing assay) Ct Ct Ct ±x times Ct Ct Ct ±x times Expression change of GOI reative to Caibrator GOI Normaized change reative to Caibrator Expression change of Normaizer reative to Caibrator Assumes Caibrator and Unknown sampes are comparabe! 9

10 WhyRNA quaitymatters Intact RNA GOI REF ΔCt for GOI and REF ΔΔCt Fod Change of GOI

11 WhyRNA QuaityMatters Degraded RNA Exampe: 50% of a REF RNAs are degraded from 5 end: GOI REF ΔCt for GOI and REF ΔΔCt Fod Change of GOI

12 Experimenta workfow Extraction from 5x10 6 HEK ces using Absoutey RNA mini RNA extraction Nuceic acid quantification and QC Quantification of 1 µ sampe on Nanodrop QC on Bioanayzer: RNA 6000 nano kit RNA 70 C RT from 1 µg of tota RNA using AffinityScript Reverse Transcription QPCR 5 and 3 assays QPCR Assay vaidation Mx3005P Briiant II SYBR Green Anaysis of QPCR products on Bioanayzer: DNA 1000 kit 12

13 Experimenta Design Look at the infuence of RNA quaity on interpretation of QPCR resuts Look at endogenous transcripts of varying expression eve Use 5 and 3 specific assays Use oigo dt and random hexamers Intentiona degradation of RNA Iustrate the vaue of the BioAnayzer in evauation of PCR products post reaction Interpretation of non-specific products 13

14 Assesment of RNA Integrity RNA 6000 Nano LabChip kit 18S 28S Typica first QC step during cdna or crna sampe prep for QPCR marker High quaity tota RNA (RIN 8.8) 2100 bioanayzer: singe ane ge-ike image 2100 bioanayzer: eectropherogram marker Partiay degraded tota RNA (RIN 3.7) 14

15 Assessment of RNA Integrity 45 Fuorescence Fuorescence S 18S 28S 28S Intact RNA: RIN 10 Partiay degraded RNA: RIN 5 Fuorescence Time (seconds) Strongy Degraded RNA: RIN 3 *Feige, S.; Pfaff, M (2006) Moecuar Aspects of Medicine 27:

16 Assesment of RNA Integrity RNA Integrity Number (RIN) RIN avaiabe for eukaryotic RNA ony!! What the RIN can do: Obtain an assessment of RNA integrity. Directy compare RNA sampes Ensure repeatabiity of experiments What it CANNOT do: Predict the outcome of an experiment if no prior vaidation was done 16

17 Assay: RNA Integrity 28S marker 18S Tota RNA was purified from 5x106 HEK ces and anayzed on the 2100 bioanayzer. The vaue of 10 obtained for the RIN shows the very high integrity of the purified sampe. 17

18 Assay: Design GAPDH: Gyceradehyde-3-phosphate dehydrogenase (Chr. 12, 9 exons) HPRT1: Hypoxanthine-guanine phosphoribosy transferase (Chr. X, 9 exons) YWHAZ: Protein kinase C inhibitor protein 1 (Chr. 8, 6 exons) intron 2-3: 23.6 kb 18

19 Assay: Vaidation BioAnayzer Discordant resuts with SYBR Met NTC NTC bp Size: 120 bp A assays had cear nort contros both for QPCR Reference RNA and HEK tota RNA no gdna present (YWHAZ 3 assay is in one exon!) One NTC was positive for the HPRT1 5 assay (167 bp): 19

20 Assay: Vaidation Beyond Meting Curve Anaysis BioAnayzer Why? SYBR Green is a nonsaturating dye Meting temperatures of products can be miseading based upon ength and sequence BioAnayzer anaysis wi give accurate high resoution information about what is being made 20

21 Assay: Vaidation BioAnayzer Concordant resuts with SYBR Met GAPDH 5 assay: Expected size 118 bp oigo-dt random HPRT1 3 assay: Expected size 114 bp oigo-dt random Size: 110 bp Size: 102 bp 21

22 Assay: Vaidation Rea-time quantitative PCR HPRT1 5 assay: Expected size 130 bp oigo-dt random HPRT1 3 assay: Expected size 114 bp oigo-dt random Size: 120 bp Size: 102 bp 22

23 Assay: Vaidation Rea-time quantitative PCR YWHAZ 5 assay: Expected size 142 bp oigo-dt random YWHAZ 3 assay: Expected size 128 bp oigo-dt random Size: 125 bp Size: 116 bp 23

24 RNA Quaity and Expression Leves A RIN 8.9 B RIN min 30 min G A P D H RIN 8.9 RIN 6.5 RIN 4.6 RIN 2.3 GAPDH 5' assay GAPDH 3' assay C RIN 4.6 D RIN min 75 min H P R T RIN 8.9 RIN 6.5 RIN 4.6 RIN 2.3 HPRT1 5' assay HPRT1 3' assay Y W H A Z RIN 8.9 RIN 6.5 RIN 4.6 RIN 2.3 YWHAZ 5' assay YWHAZ 3' assay 24

25 Summary DNA/RNA quaity can have a dramatic effect on QPCR resuts Performing quantification assumes comparabe tempate quaity Extent of effect is sequence/gene dependent For successfu QPCR it is advisabe to optimize sampe preparation methods to achieve highest tempate quaity possibe Adjust design of ampicon (position/size) based on sampe quaity The Bioanayzer aows easy assessment of RNA quaity and faciitates QPCR assay vaidation DNA fragment anaysis on Bioanayzer more sensitive as SYBR Green metcurve 25

26 Additiona Information 26

27 How can the BioAnayzer improve QPCR gene expression Anaysis? 27

28 Gene Expression Workfow Absoutey RNA RNA extraction Nuceic acid quantification and QC AffinityScript Reverse Transcription QPCR Assay vaidation QPCR 28

29 Miniaturization (Scae) sma sampe voumes reduced reagent usage reduced bench space Bioanayzer Lab-on-a-Chip Genera Features and Benefits Miniaturization (Speed) fast anaysis 01:30:00 00:00:15 00:00:15 Automation improved accuracy improved precision improved productivity Smaer - Faster - Smarter from sampe to digita data - quicky and reproduciby 29

30 Lab-on-a-Chip Technoogy An Overview Based on Microfuidics the movement of iquids through micro-fabricated structures by means of eectrica fieds or pressure/vacuum sma gass or pastic devices with micro-channes as experimenta patform active contro of fuids without moving parts on-chip through miniature eectrodes or pumps controed by software scripts emuation of conventiona iquid pumps, vaves, dispensers, reactors, separation systems, etc. capabiity of iquid transfer, separation, diution, reactions and more hoding the promise of greater functionaity with significanty improved reiabiity! 30

31 Lab-on-a-Chip or Ge 15 min 25 min 40 min Prep Chip Run Chip Anayzed - Archived data 2 hours- 1 day 15 min 30 min-2 hours 30 min-8 hours 30 min Prep Ge Run Ge Stain / Destain Scan / Anayze 31

32 2100 Bioanayzer Hardware Bayonet Cartridge Exchangeabe cartridge for eectrophoresis or fow cytometry assays 16 pin eectrodes connected to HV-sources Chip hoder with heater pate Chip seector Optics for detection 32

33 Chip for Moecuar Assays Anaysis of DNA, RNA and proteins. DNA chip dispayed as exampe. Sampe wes Ge wes Ge wes Ladder we Separation channe and point of detection Ladder we Chip accommodates sampe wes, ge wes and a we for a standard (adder) 16 pin-eectrodes in the eectrode cartridge (standard equipment) are arranged such that they fit in the wes on the chip on-chip ge eectrophoresis 33

34 Principe of Eectrodriven Fow 1. The sampe moves eectrodriven from the sampe we through the micro-channes 2. The sampe is eectrokineticay injected into the separation channe 3. Sampe components are eectrophoreticay separated 4. Components are detected by their fuorescence and transated into ge-ike images (bands) and eectropherograms (peaks) The microchannes of the gass chip are fied with a sieving poymer and fuorescent dye 34

35 Principe of Eectrodriven Fow 35

36 The Lab-on-a-Chip Approach Active Contro of Fuids without Moving Parts Sampe voumes 1-4 µ sampes depending on Assay Separation, staining, detection of sampes Resuts in 5-30 minutes avaiabe No extra waste remova needed Disposabe Chip, no crosscontamination 36

37 RNA Appications RNA QA/QC for Microarrays Gene Expression RNA QA/QC for qpcr RNA QA/QC for mpcr smarna QA/QC Genomic DNA Contamination 37

38 Protein Appications Food Anaysis Antibody QA/QC Protein Expression Compiant Protein & Antibody QA/QC Protein Purification Protein QA/QC 38

39 DNA Appications mtdna Screening Forensic Testing Food Anaysis qpcr vaidation, impurity check mpcr vaidation, impurity check Cinica Research Oncoogy Restriction Digest Anaysis Gene Expression 39

40 Ce Appications Transfection Efficiency sirna Transfection Efficiency GFP Transfection Efficiency Antibody Staining Protein Expression Monitoring Apoptosis Annexin V Apoptosis Caspase-3 40

41 Introduction Current Assays DNA1000 DNA7500 DNA12000 DNA Assays: Sizing Quantitation PCR products, digests, arger DNA fragments 12 sampes in 30 min Nano 6000 Pico Sma RNA RNA Assays: Quantitation (Sizing in Sma RNA) tota RNA, mrna purity & integrity determination 10 sampes in 30 min. Fow Cytometry Ce Assays: Anaysis of 6 sampes Two coor detection Anaysis of protein expression in ces P230 P80 P250 Protein Assays: Sizing Quantitation ce ysates, coumn fractions, purified proteins, antibodies etc. 10 sampes in 40 min. 41