Analyze microrna Activity in Cells Using Luciferase Reporters. Trista Schagat, Ph.D. December 2013

Transcription

1 Analyze microrna Activity in Cells Using Luciferase Reporters Trista Schagat, Ph.D. December 2013

2 agenda microrna Overview How to and Tips Design Transfect Assay Analyze Case Studies 2

3 micrornas are highly abundant ~21nt single stranded RNAs Huntzinger and Izaurralde (2011) Nature Rev. Gen. 12:

4 mircrorna sequence archive has grown as quickly as the list of microrna-related publications >24,521 microrna genes in over 140 species >2500 mature human micrornas >60% human protein coding genes may be targets Kozomara and Griffiths-Jones (2011) Nucl. Acids Res. 39:D152-D157 Friedman et al. (2009) Gen Rsch 19:

5 microrna biogenesis Pasquinelli (2012) Nature Rev. Gen. 13:

6 micrornas decrease target expression by direct RNA interaction Pasquinelli (2012) Nature Rev. Gen. 13:

7 Reporters facilitate microrna research 7

8 Use of reporters to study micrornas is a 4 stage process Design Transfect Assay Analyze 8

9 Analyze the target for microrna sequences using open access tools Design

10 Choose region to fuse to 3 UTR of reporter Design Target 3 UTR: 5 Reporter 3 5 Target 3 UTR 3 Target microrna response element: 5 Reporter 3 MRE 5 3 Biosensor with complimentary binding site: 5 Reporter 3 MBS

11 Plasmid should include basic elements Design MCS Poly(A) signal Reporter pmirglo Vector (Cat. #E1330) Minimal promoter 11

12 Create transfection-quality plasmid DNA stocks Design PureYield Plasmid Midiprep System (Cat. #A2495) Endotoxin Removal Wash 12

13 Use of reporters to study micrornas is a 4 stage process Design Transfect Assay Analyze Target element(s) of interest Simple vector design Prepare quality DNA stocks 13

14 Keep cells healthy to preserve biology Transfect X Visually monitor cells Monitor passage number Do not transfect over confluent cells Cells used for seeding of transfection plates should be sub confluent 14

15 Optimize transfections for high expression and low cell death Transfect Transfection: FuGENE 6 (Cat. #E2691) FuGENE HD (Cat. #E2311) ONE-Glo + Tox Luciferase Reporter and Cell Viability Assay (Cat. #E7110) 15

16 Optimize transfection conditions first with single reporter, then optimize for multiple inputs Transfect Optimize Round 1 Round 2 Cell culture variables cell density, medium, exposure Reagent variables ratio, total mass DNA, volume Dual Reporter ratios 20:1, 10:1, 1:1, 1:10, 1:20 Effector expression plasmid Putative co-regulatory component(s) Regulatory Oligos mirna mimic, antimirs 16

17 Internal vector control corrects for transfections and non-critical variables Transfect Schagat, T. et al. (2007) Cell Notes 17:

18 Cotransfect oligonucleotides to manipulate microrna function Transfect Small and Olson (2011) Nature 469 (7330),

19 Use of reporters to study micrornas is a 4 stage process Design Transfect Assay Analyze Keep cells healthy to preserve biology Optimize for cells and reagent Optimize for multiple plasmids and oligos 19

20 Target regulation by micrornas can be assayed by reporter protein and mrna Assay 20

21 Luciferase reporter protein assays are easy and fast Assay Add Dual-Glo Luciferase Assay Reagent Dual-Glo Luciferase Assay System (Cat. #E2920) Incubate 10 min. Measure firefly luminescence Add Dual-Glo Stop & Glo Reagent Incubate 10 min. Measure Renilla luminescence 21

22 Reporter RNA assays are multi-step and must minimize plasmid DNA carryover Assay RNA Extraction ReliaPrep RNA Miniprep Systems* Maxwell 16 LEV simplyrna Kits* DNase Treatment RQ1 RNase-Free DNase RT-qPCR Plexor qpcr Systems GoTaq qpcr Systems *protocol modification needed for cytoplasmic RNA 22

23 Controls are critical Assay Control No (or mock) transfection control Internal transfection control Vehicle control (if treating cells) No Template Control No RT control Reporter Protein Assay Establish background Reporter mrna RTqPCR Assay Correct for well to well variation in transfections Monitor for non-specific effects NA NA Environmental contamination Plasmid DNA contamination 23

24 Use of reporters to study micrornas is a 4 stage process Design Transfect Assay Analyze Simple luc protein assay Minimize plasmid in RNA Include controls 24

25 Analyze reporter protein assay data Analyze Raw data Analyze Normalize for internal control Calculations Average the background for each reporter Background subtract from each sample and reporter Calculate average ± std dev of replicates Calculate Relative Response Ratio for each RRR luc /RRR internal control Average ± std dev of replicates Percent repression [1 (RRR MRE /mean RRR mutmre )] x

26 Carefully examine data throughout analysis to identify issues Analyze Schagat, T. et al. (2013) Methods 63,

27 Pfaffl method can be used to analyze RT-qPCR data Analyze Analyze ΔCt correct for internal control Calculations Ct test reporter Ct internal control ΔΔCt Compare to unregulated control Percent repression Determine magnitude of effect ΔCt MRE mean ΔCt mutmre [1 (2 -ΔΔCt )] x 100 Pfaffl (2001) Nucl. Acids Rsch. 29, e45. 27

28 Example reporter RNA data analysis Analyze Schagat, T. et al. (2013) Methods 63,

29 Use of reporters to study micrornas is a 4 stage process Design Transfect Assay Analyze Protein & RNA similar Normalized data reduces variability Look for issues 29

30 Case Studies for microrna analysis with reporters 30

31 Case Study #1 uses bioinformatics to ID mir sites then reporters to study regulation 5 Reporter 3 Target 3 UTR 5 3 Wang, Z. and Burke, P.A. (2013) Biochim et Biophys Acta 1829,

32 Case Study #1 uses bioinformatics to ID mir sites then reporters to study regulation Wang, Z. and Burke, P.A. (2013) Biochim et Biophys Acta 1829,

33 Case Study #2 describes a novel bioinformatics tool and uses reporters to validate predictions 5 Reporter 3 Target 3 UTR 5 3 Plaisier, C.L. et al. (2012) Genome Res. 22,

34 Case Study #2 describes a novel bioinformatics tool and uses reporters to validate predictions Plaisier, C.L. et al. (2012) Genome Res. 22,

35 Case Study #3 uses reporters to screen for smallmolecule inhibitors of microrna function 5 Reporter 3 MRE 5 3 Connelly C. M. et al. (2012) J. Biomol. Screen 17,

36 Case Study #3 uses reporters to screen for smallmolecule inhibitors of microrna function Connelly C. M. et al. (2012) J. Biomol. Screen 17,

37 Use reporters to study microrna activity in cells Design Transfect Assay Analyze Target, vector quality DNA Healthy cells Optimize Simple luc assay plasmid in RNA Controls Normalized data Look for issues 37

38 Publication describes process in detail with ready to use protocols A guide to design and optimization of reporter assays for 3 untranslated region mediated regulation of mammalian messenger RNAs. Schagat, T. et al. (2013) Methods 63,

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