MicroRNA Analysis Paired with Novel Cell Health Assays: A Complete Workflow Brad Hook, Ph.D Manager, NA Scientific Applications 2016
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 2016. 2
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 2016. 3
Study 1: mirnas Associated with Cancer 2016. 4
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) 2016. mrna/microrna profiling Amplification 5
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. 2016. 6
Cells in Culture 2D vs 3D Many 3D models exist. In this experiment we used: 3D Spheroids Matrigel ~700µm 2016. 7
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 2016. 8
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 2016. 9
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 2016. 10
Why Use Real-Time Assays? Benefits of multiplexing and kinetic monitoring: Saves $, time, plastics Fewer cells More data from a single sample Data normalization 2016. 11
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 2016. 12
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 140 2.50 120 2.30 100 2.10 80 1.90 1.70 60 1.50 40 1.30 20 1.10 0 0.90-6.5-6 -5.5-5 -4.5-4 -3.5 Log[5-Fluorouracil], M HT-29 1hr Viability 24hr Viability 48hr Viability 48hr Cytotoxicity 140 2.50 120 2.30 100 2.10 80 1.90 1.70 60 1.50 40 1.30 20 1.10 0 0.90-6.5-6 -5.5-5 -4.5-4 -3.5 Log[5-Fluorouracil], M 2016. 13
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 2016. 14
Percent viability Fold change in cytotoxicity Study 1: Potency Over Time Why 30µM 5-Fluorouracil? 1hr Viability 48hr Viability HCT116 24hr Viability 48hr Cytotoxicity 140 120 2.40 100 80 1.90 60 40 1.40 20 0 0.90-6.5-6 -5.5-5 -4.5-4 -3.5 Log[5-Fluorouracil], M 2016. 15
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 2016. mrna/microrna profiling Amplification 16
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 2016. 17
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? 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 HCT116-Caspase-Glo 3/7 3D Matrigel 3D Spheroid 2D Conclusion: Cells are not going through apoptosis. Why not? 2016. 18
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 140 120 2.40 100 80 1.90 60 40 1.40 20 0 0.90-6.5-6 -5.5-5 -4.5-4 -3.5 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. 2016. 19
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 2016. mrna/microrna profiling Amplification 20
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 2016. mrna/microrna profiling Amplification 21
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 2016. 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. ReliaPrep mirna Cell and Tissue Miniprep System Fast and simple less than 40min Isolates total RNA including mirna No phenol:chloroform No ethanol precipitation 2016. 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. 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. 2016. 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. Total RNA isolation mrna/microrna profiling Amplification HCT116 5-Fluorouracil 30µM 48hr Taqman mirna Reverse Transcription kit + GoTaq Probe qpcr Master Mix 2016. 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. 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 2016. 26
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 2016. mrna/microrna profiling Amplification 27
Fold Change from DMSO control Study 1: Pulling It All Together 1.4 1.2 1 0.8 0.6 0.4 0.2 0 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. 2016. 28
Yield (ng) Study 1: Pulling It All Together 1000 900 800 700 600 500 400 300 200 100 0 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. 2016. 29
Study 1: Pulling It All Together mir-302a-3p 2D 3D spheroid 3D Matrigel mir-323-3p mir-320c mir-30b-5p 0.1 1 10 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. 2016. 30
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 2016. 31
Study 2: Experimental Workflow MicroRNA Analysis Paired with a Novel Live Cell Viability Assay: A Complete Epigenetic Workflow in Human Cancer Cell Lines 2016. 32
Study 2: Experimental Workflow MicroRNA Analysis Paired with a Novel Live Cell Viability Assay: A Complete Epigenetic Workflow in Human Cancer Cell Lines 2016. 33
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 2016. 34
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 231 2 MCF7 MDA MB 231 1 1.5 0.5 0 1µM TMP269 2mM Sodium Butyrate DMSO 1 0.5 0 1µM TMP269 2mM Sodium Butyrate DMSO HDAC activity decreases with TMP269 and Sodium Butyrate treatment. 2016. 35
Study 2: Experimental Workflow MicroRNA Analysis Paired with a Novel Live Cell Viability Assay: A Complete Epigenetic Workflow in Human Cancer Cell Lines 2016. 36
% 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 120 100 80 60 40 20 0 MCF7 4 24 48 1µM TMP269 2mM Sodium Butyrate 140 120 100 80 60 40 20 0 MDA-MB-231 4 24 48 1µM 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. 2016. 37
% 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. 150 100 50 Cytotoxicity monitored during HDAC inhibitor treatment MCF7 4 24 48 150 100 50 MDA-MB-231 4 24 48 0 1µM 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. 2016. 38
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 2016. 39
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 2016. 40
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. 1.2 1 0.8 0.6 0.4 0.2 0 mrna Expression (CDK2) 1µM TMP269 2016. 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 5 4 3 2 1 0-1 mirna Expression MCF7 MDA MB 231 1µM TMP269 2 mm Sodium Butyrate 41
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 2016. 42
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 2016. 43
Technical Services Scientists Ready to Help 2016. 44
Thank you for attending and we would be happy to answer any questions you may have in the chat window. Learn more about the Promega products used in this webinar: RealTime-Glo MT Cell Viability Assay CellTox Green Cytotoxicity Assay Caspase-Glo 3/7 Assay HDAC-Glo I/II Assay HDAC-Glo Class IIa Assay QuantiFluor RNA System Quantus Fluorometer GloMax Discover ReliaPrep mirna Cell and Tissue Miniprep System GoTaq Probe qpcr Master Mix GoTaq Probe 1-Step RT-qPCR System 2016. 45