Slide 1Differentiation of HL-60 CellsSlide 3Control of genetic expressionSlide 5Chapter 5: RT-PCRChapter 5: RT-PCRRNA isolationSlide 9cDNA (complementary DNA) vs. gDNA (genomic DNA)Polymerase Chain Reaction (PCR)Typical PCR programWhat goes in the Polymerase Chain Reaction (PCR)?MMP-9 gene expressionβ-actin gene expressionReview of our experiment:Controls in today’s experimentHow do we view our PCR results?Agarose Gel ElectrophoresisAgaroseGel ElectrophoresisGel Electrophoresis AnimationChapter 5Differentiation of HL-60 Cells•Cell becomes more specialized, takes on new function•Can cause changse in:–Morphology–Gene expression (does a gene get transcribed?)–Protein production (does mRNA get translated?)•Last week we stained the cells using May-Grünwald/Giemsa to view:MORPHOLOGICAL CHANGES•This week we will isolate RNA to view:TRANSCRIPTIONAL CHANGEShttp://www.dnalc.org/resources/3d/13-transcription-advanced.htmlhttp://www.dnalc.org/resources/3d/16-translation-advanced.htmlCentral dogma of molecular biologyThe principle of directional flow of genetic information proceeds from:DNADNA replication•transcription (copying information without “language” change) from DNA to RNA•translation (language change from nucleotide sequence to amino acid sequence) from RNA to protein. ProteinRNAtranslationtranscriptionControl of genetic expression•This occurs at five levels;–Genomic level (is the DNA even accessible for transcription; chromatin?)–During transcription (Is the gene actively transcribed or not; that is, is mRNA produced?)–RNA processing (5’ capping, RNA Splicing (removal of introns; joining of exons for functional mRNA, 3’ polyadenylation of mRNA)–During translation (Does the mRNA get translated into protein?)–Post translational i.e. proMMP-9  MMP-9PMA Signal Transduction PathwayIn the presence of PMA a signal is triggered via receptors in the cell membrane to a secondary messenger, PKC-β which in turn induces a response in the DNA for the cell to modify and produce the end product, MMP-9Experimental focus:Gene expressionChapter 5: RT-PCR•You will examine HL-60 cells that have been treated with PMA or DMSO.•We will perform Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) to examine changes in gene expression, specifically the transcription of the MMP-9 gene in response to HL-60 cell differentiation into monocytes with PMA treatment–We are looking at the mRNA production! (remember that mRNA is translated into protein)Chapter 5: RT-PCR-Reverse-Transcriptase Polymerase Chain Reaction -RNA isolation: RNA is isolated from the cells. (Exercise 5.1)-Reverse transcription: The RNA is converted to complementary DNA (cDNA) (Exercise 5.2)-Polymerase Chain Reaction: Primers specific to a gene of interest are used to exponentially amplify a segment of the gene of interest. (Exercise 5.3)-The PCR products are viewed and analyzed using agarose gel electrophoresis.RNA isolationRemember - RNA is VERY unstable!•Single stranded , making it susceptible to Ribonucleases (Rnases). •RNases are everywhere, so all materials must be sterile (autoclaved)•2’ -OH configuration of backbone, makes RNA susceptible to hydrolysisSugar Phosphate BackboneNitrogenous Bases – AUCG’sOHPhosphateSugarBaseReverse Transcription: The process of converting mRNA into cDNA http://8e.devbio.com/images/ch04/0405fig2.jpgEnzyme that carries out this reaction is Reverse TranscriptasecDNA (complementary DNA) vs. gDNA (genomic DNA)gDNA; total DNA from cellsmRNARNA processing!TranscriptionReverse TranscriptioncDNA; representative of gDNA actually transcribed into mRNA(recall more stable than mRNA)Polymerase Chain Reaction (PCR)•Used to amplify a certain gene of interest•Three steps:–1. Denaturing of DNA–2. Annealing of primers–3. Extension of DNA•Results in exponential amplification of your target gene•See animation:http://www.dnalc.org/resources/3d/19-polymerase-chain-reaction.htmlTypical PCR program1. 95°C for 5 min (Denature)2. 95°C, 1min (Denature)3. 55°C, 1min (Anneal primers)4. 72°C, 1min (Extension of primers)5. 72°C, 5min (Extension) **NOTE: this is a general template program, all times and temperatures can be changed to accommodate your DNA and primer conditionsWhat goes in the Polymerase Chain Reaction (PCR)?•Taq polymerase; a heat stable DNA polymerase•Deoxynucleotides ; dATP, dGTP, dTTP, dCTP.•Primers - Short oligomers/oligos designed to target a segment of your gene of interest; two sets of primers will be run on your samples–1 set targeting a segment of the MMP-9 gene–1 set targeting a segment of the β-actin gene.•β-actin is a housekeeping gene, therefore very abundant in all cells, and will be used as a control to verify that your reverse transcription worked properly–Template DNA; for this experiment, it’s your cDNA from PMA-treated and DMSO-treated HL-60 cells.MMP-9 gene expressionBy reverse transcription, we will convert mRNA including the MMP-9 mRNA into cDNA and then use PCR to amplify the cDNA.Monocytes express the MMP-9 gene; which means that the MMP-9 gene is actively transcribed, MMP-9 mRNA present!PMA treatment induces HL-60 cells to differentiate into monocytesBy reverse transcription, we will convert mRNA into cDNA and then use PCR to amplify the cDNA.(* Some MMP-9 mRNA present due to spontaneous differentiation into monocytes)Granulocytes do not express the MMP-9 gene; which means that the MMP-9 gene is not actively transcribed, MMP-9 mRNA absent!DMSO treatment induces HL-60 cells to differentiate into granulocytesβ-actin gene expressionBy reverse transcription, we will convert mRNA including the β-actin mRNA into cDNA and then use PCR to amplify the cDNA.Monocytes express β-actin gene gene; which means that the β-actin gene is actively transcribed, β-actin mRNA present!The β-actin gene is a housekeeping gene, constitutively expressed regardless of PMA treatment! By reverse transcription, we will convert mRNA including the β-actin mRNA into cDNA and then use PCR to amplify the cDNA.Granulocytes express β-actin gene; which means that the β-actin gene is actively transcribed, β-actin mRNA present!The β-actin gene is a housekeeping gene, constitutively expressed regardless of DMSO treatment!Review of our experiment:1. Measure cell density and cell viability using a Trypan Blue Assay2. Isolate RNA from PMA/DMSO treated HL-60 cells3. Reverse Transcribe RNA to cDNAThis is where you will stop