MicroRNA Analysis Paired with Novel Cell Health Assays: A Complete Workflow
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1 MicroRNA Analysis Paired with Novel Cell Health Assays: A Complete Workflow Brad Hook, Ph.D Manager, NA Scientific Applications 2016
2 Presentation Outline From cells to RNA a seemingly easy, yet complex path Study 1: Differential expression of mirna in 2D and 3D human colon cancer cell cultures after therapeutic compound treatment Study 2: MicroRNA analysis paired with a novel live cell viability assay: A complete epigenetic workflow in human cancer cell lines
3 From Cells to RNA Profiling Complete Workflow Cells Compound Viability Total RNA Purification Incubation Quantitation Amplification Cytotoxicity Other Cell-Based Tests: Enzyme activity HDAC P450 assay Caspase activity Many others
4 Study 1: mirnas Associated with Cancer
5 Study 1: Experimental Workflow Differential expression of mirna in 2D and 3D human colon cancer cell cultures after therapeutic compound treatment Live cells Dead cells Apoptotic cells Caspase activity Live cell 2 Necrosis Compound Treatment (5-Fluorouracil) mrna/microrna profiling Amplification 5
6 Cells in Culture 2D vs 3D Why 3D cell culture? Cells growing on surfaces are artificial and unnatural Extra-cellular matrix plays an important role in regulating cellular behavior by influencing cells with biochemical signals and topographical cues In 3D culture, we can control scaffold morphology, architecture and components Therefore cells behave and respond more like they would in vivo to stimuli
7 Cells in Culture 2D vs 3D Many 3D models exist. In this experiment we used: 3D Spheroids Matrigel ~700µm
8 Study 1: Experimental Setup Part 1: Determine compound potency at multiple time-points in two human colon cancer cell lines, HCT116 and HT-29. Live cells Dead cells Cell Health Compound Treatment (5-Fluorouracil) 48hr
9 Study 1: Cell Viability RealTime-Glo Part 1: Determine compound potency at multiple time-points in two human colon cancer cell lines, HCT116 and HT-29. RealTime-Glo MT Cell Viability Assay Live cells Dead cells Cell Health Compound Treatment (5-Fluorouracil) 48hr
10 Study 1: Cytotoxicity CellTox Green Part 1: Determine compound potency at multiple time-points in two human colon cancer cell lines, HCT116 and HT-29. CellTox Green Cytotoxicity Assay Live cells Dead cells Cell Health Compound Treatment (5-Fluorouracil) 48hr
11 Why Use Real-Time Assays? Benefits of multiplexing and kinetic monitoring: Saves $, time, plastics Fewer cells More data from a single sample Data normalization
12 Essential Equipment Plate Reader GloMax Discover Multimode Detection System Luminescence, Fluorescence, and Absorbance (UV-Visible) 6-, 12-, 24-, 48-, 96- and 384-well formats Filtered Luminescence, BRET, FRET Easy to use software Integrated protocols Customizable protocols
13 Percent viability Fold change in cytotoxicity Percent viability Fold change in cytotoxicity Study 1: Potency Over Time Two human colon cancer cell lines (2D cultures) were treated with 5-Fluorouracil and monitored for viability and cytotoxicity over a 48 hour time period using the RealTime-Glo MT Cell Viability and CellTox Green Cytotoxicity Assays. HCT116 1hr Viability 24hr Viability 48hr Viability 48hr Cytotoxicity Log[5-Fluorouracil], M HT-29 1hr Viability 24hr Viability 48hr Viability 48hr Cytotoxicity Log[5-Fluorouracil], M
14 Study 1: Potency Over Time Part 1: Conclusion A dose-dependent response to 5-Fluorouracil was observed in the HCT116 cells. As compound dose increased, viability decreased and cytotoxicity increased. As exposure to 5-Fluorouracil increased, cell viability decreased. HT-29 cells were more resistant to 5-Fluorouracil compared to HCT116 cells. Live cells Dead cells Cell Health Compound Treatment (30µM) HCT116 48hr
15 Percent viability Fold change in cytotoxicity Study 1: Potency Over Time Why 30µM 5-Fluorouracil? 1hr Viability 48hr Viability HCT116 24hr Viability 48hr Cytotoxicity Log[5-Fluorouracil], M
16 Study 1: Experimental Workflow Differential expression of mirna in 2D and 3D human colon cancer cell cultures after therapeutic compound treatment Used to determine potency and timing Live cells Dead cells Apoptotic cells Caspase activity Live cell 2 Necrosis HCT116 5-Fluorouracil - 30µM 48hr mrna/microrna profiling Amplification 16
17 Study 1: Apoptosis Part 2: Determine mode of death are cells starting to become apoptotic with 30µM treatment of 5-Fluorouracil at 48hr? Used to determine mode of death Caspase activity Apoptotic cells Live cell 2 Necrosis HCT116 5-Fluorouracil - 30µM 48hr Caspase-Glo 3/7 Assay
18 Fold change from DSMO control Study 1: Apoptosis Part 2: Determine mode of death are cells starting to become apoptotic with 30µM treatment of 5-Fluorouracil at 48hr? HCT116-Caspase-Glo 3/7 3D Matrigel 3D Spheroid 2D Conclusion: Cells are not going through apoptosis. Why not?
19 Percent viability Fold change in cytotoxicity Study 1: Apoptosis Part 2: Determine mode of death are cells starting to become apoptotic with 30µM treatment of 5-Fluorouracil at 48hr? 1hr Viability 48hr Viability HCT116 24hr Viability 48hr Cytotoxicity Log[5-Fluorouracil], M Cells are not dying at this time point with this dose of 5-Fluorouracil. We might observe apoptosis at longer time points and higher doses of compound
20 Study 1: Experimental Workflow Part 2: Determine mode of death are cells starting to become apoptotic with 30µM treatment of 5-Fluorouracil at 48hr? Live cells Dead cells Cells not going through apoptosis Apoptotic cells Caspase activity Live cell 2 Necrosis HCT116 5-Fluorouracil 30µM 48hr mrna/microrna profiling Amplification 20
21 Study 1: Experimental Workflow Part 3: mrna/microrna profiling. Determine changes in mrna and mirna transcript levels after 30µM treatment of 5-Fluorouracil at 48hr. Live cells Dead cells Apoptotic cells Caspase activity Live cell 2 Necrosis HCT116 5-Fluorouracil 30µM 48hr mrna/microrna profiling Amplification 21
22 Study 1: mrna/microrna Analysis Part 3: mrna/microrna profiling. Determine changes in mrna and mirna transcript levels after 30µM treatment of 5-Fluorouracil at 48hr. mrna/microrna profiling Total RNA isolation Amplification HCT116 5-Fluorouracil 30µM 48hr ReliaPrep mirna Cell and Tissue Miniprep System
23 Study 1: mrna/microrna Analysis Part 3: mrna/microrna profiling. Determine changes in mrna and mirna transcript levels after 30µM treatment of 5-Fluorouracil at 48hr. ReliaPrep mirna Cell and Tissue Miniprep System Fast and simple less than 40min Isolates total RNA including mirna No phenol:chloroform No ethanol precipitation
24 Study 1: mrna/microrna Analysis Part 3: mrna/microrna profiling. Determine changes in mrna and mirna transcript levels after 30µM treatment of 5-Fluorouracil at 48hr. Before RT-qPCR, it is important to quantify the RNA levels in the sample eluates. The QuantiFluor RNA system contains a fluorescent RNA-binding dye that enables sensitive quantitation of small amounts of RNA in solution. The Quantus fluorometer is a small, dual-channel fluorometer for providing highly sensitive fluorescent detection when quantifying nucleic acids
25 Study 1: mrna/microrna Analysis Part 3: mrna/microrna profiling. Determine changes in mrna and mirna transcript levels after 30µM treatment of 5-Fluorouracil at 48hr. Total RNA isolation mrna/microrna profiling Amplification HCT116 5-Fluorouracil 30µM 48hr Taqman mirna Reverse Transcription kit + GoTaq Probe qpcr Master Mix
26 Study 1: mrna/microrna Analysis Part 3: mrna/microrna profiling. Determine changes in mrna and mirna transcript levels after 30µM treatment of 5-Fluorouracil at 48hr. Taqman mirna Reverse Transcription kit + GoTaq Probe qpcr Master Mix Expected results based on literature mir-302a-3p mir-323-3p mir-320c mir-30b-5p RNU6B
27 Study 1: Pulling It All Together Part 3: mrna/microrna profiling. Determine changes in mrna and mirna transcript levels after 30µM treatment of 5-Fluorouracil at 48hr. Live cells Dead cells HCT116 5-Fluorouracil - 30µM 48hr mrna/microrna profiling Amplification 27
28 Fold Change from DMSO control Study 1: Pulling It All Together Viability Cell Health Assays Control 3D Matrigel 3D Spheroid 2D Cytotoxicity Cells (all cultures) treated with 30µM 5-Fluorouracil showed a decrease in viability but no significant increase in cytotoxicity, indicating an anti-proliferative effect
29 Yield (ng) Study 1: Pulling It All Together Total RNA Yield 5-FU DMSO 5-FU DMSO 5-FU DMSO 3D Matrigel 3D spheroid 2D Total RNA was isolated from all samples and quantitated using the QuantiFluor RNA System on the Quantus Fluorometer. As expected RNA yields were lower with treated samples as the number of viable cells were lower than the DMSO control
30 Study 1: Pulling It All Together mir-302a-3p 2D 3D spheroid 3D Matrigel mir-323-3p mir-320c mir-30b-5p Fold Change in mirna expression With all four mirnas tested, the fold change in mirna expression was lowest with 3D spheroids, highest with 2D cultures and moderate with 3D Matrigel. This could be due to the inability of the compound to penetrate to the center of larger 3D complexes or the difference in culturing format effects on cellular responsiveness
31 Study 2: Experimental Workflow Compare two common breast cancer cell lines for responses to HDAC inhibitor treatment Breast Adenocarcinoma Cell Lines Tested: MCF7: HER low, ERα+, PR+ MDA-MB-231: Triple Negative HDAC Inhibitors TMP269: HDAC class IIa Inhibitor Sodium Butyrate: Pan HDAC Inhibitor
32 Study 2: Experimental Workflow MicroRNA Analysis Paired with a Novel Live Cell Viability Assay: A Complete Epigenetic Workflow in Human Cancer Cell Lines
33 Study 2: Experimental Workflow MicroRNA Analysis Paired with a Novel Live Cell Viability Assay: A Complete Epigenetic Workflow in Human Cancer Cell Lines
34 Study 2: HDAC Activity Part 1: Test HDAC activity in MCF7 and MDA-MB-231 cells when treated with Sodium Butyrate and TMP269 for 48hr. Sodium Butyrate and TMP269 are known HDAC inhibitors. Based on data from previous studies, we chose a single dosing concentration of each compound (2mM Sodium Butyrate and 1µM TM269) known to effect HDAC activity. Promega offers multiple HDAC assays: HDAC-Glo I/II Assay HDAC-Glo Class IIa HDAC-Glo 2 SIRT-Glo Assays
35 Relative Activity to Control Relative Activity to DMSO Study 2: HDAC Activity Part 1: Test HDAC activity in MCF7 and MDA-MB-231 cells when treated with Sodium Butyrate and TMP269 for 48hrs. HDAC-Glo I/II Assay HDAC-Glo Class IIa Assay 1.5 MCF7 MDA MB MCF7 MDA MB µM TMP269 2mM Sodium Butyrate DMSO µM TMP269 2mM Sodium Butyrate DMSO HDAC activity decreases with TMP269 and Sodium Butyrate treatment
36 Study 2: Experimental Workflow MicroRNA Analysis Paired with a Novel Live Cell Viability Assay: A Complete Epigenetic Workflow in Human Cancer Cell Lines
37 % Viability Relative to DMSO % Viability Relative to DMSO Study 2: Cell Health Assays Part 2: Determine viability and toxicity of MCF7 and MDA-MB-231 cells when treated with Sodium Butyrate and TMP269 for 48hr. Cell viability monitored during HDAC inhibitor treatment MCF µM TMP269 2mM Sodium Butyrate MDA-MB µM TMP269 2mM Sodium Butyrate Cell viability was measured with RealTime-Glo MT Cell Viability Assay at 4, 24, and 48 hours after compound treatment. N=8 for each condition
38 % Signal Relative to DMSO % Signal Relative to DMSO Study 2: Cell Health Assays Part 2: Determine viability and toxicity of MCF7 and MDA-MB-231 cells when treated with Sodium Butyrate and TMP269 for 48hrs Cytotoxicity monitored during HDAC inhibitor treatment MCF MDA-MB µM TMP269 2mM Sodium Butyrate 0 1µM TMP269 2mM Sodium Butyrate Cell death was measured with CellTox Green Cytotoxicity Assay at 4, 24, and 48 hours after compound treatment. N=8 for each condition
39 Study 2: Cell Health Assays Part 2: Determine viability and toxicity of MCF7 and MDA-MB-231 cells when treated with Sodium Butyrate and TMP269 for 48hr. MCF7 : TMP269 No Effect on Viability No Effect on Cell Death No Effect on HDAC I/II activity Decrease in HDAC Class IIa Activity Sodium Butyrate Decrease in Cell Growth Minimal increase in Cell Death Decrease in HDAC I/II activity Increase in HDAC Class IIa Activity MDA-MB-231 : TMP269 No Effect on Viability No Effect on Cell Death No Effect on HDAC I/II activity Decrease in HDAC Class IIa Activity Sodium Butyrate Minimal Decrease in Growth Minimal increase in Cell Death Decrease in HDAC I/II activity No Effect on HDAC Class IIa Activity
40 Study 2: Experimental Workflow MicroRNA Analysis Paired with a Novel Live Cell Viability Assay: A Complete Epigenetic Workflow in Human Cancer Cell Lines ReliaPrep mirna Cell and Tissue Miniprep System
41 mir31 mir141 mir20a mir31 mir141 mir20a Relative Expression Relative Expression Study 2: mrna/mirna Analysis Part 3: mrna and mirna expression levels in MCF7 and MDA-MB-231 cells when treated with Sodium Butyrate and TMP269 for 48hr mrna Expression (CDK2) 1µM TMP MCF7 MDA MB 231 2mM Sodium Butyrate DMSO CDK2 mrna expression is decreased with sodium butyrate treatment in both cell lines mir31 expression is increased with sodium butyrate treatment in MCF7 cells mirna Expression MCF7 MDA MB 231 1µM TMP269 2 mm Sodium Butyrate 41
42 Study 2: Conclusions MCF7 : TMP269 No Effect on Viability No Effect on Cell Death No Effect on HDAC I/II activity Decrease in HDAC Class IIa Activity Sodium Butyrate Decrease in Cell Growth Minimal increase in Cell Death Decrease in HDAC I/II activity Increase in HDAC Class IIa Activity Decrease in CDK2 mrna Decrease in mir31 MDA-MB-231 : TMP269 No Effect on Viability No Effect on Cell Death No Effect on HDAC I/II activity Decrease in HDAC Class IIa Activity Sodium Butyrate Minimal Decrease in Growth Minimal increase in Cell Death Decrease in HDAC I/II activity No Effect on HDAC Class IIa Activity Decrease in CDK2 mrna
43 From Cells to RNA Profiling Complete Workflow Cells Compound Viability Total RNA Purification Incubation Quantitation Amplification Cytotoxicity Other Cell-Based Tests: Enzyme activity HDAC P450 assay Caspase activity Many others
44 Technical Services Scientists Ready to Help
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