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Lecture 7 • Assignment 2 • RT-PCR process and principles • Etiolation • DET1 • Discussion of Article 4 Assignment 2 • • • • • • • Sample 1 General Hans Canis Sample 2 Field Marshall Heinrich Rattus Sample 3 Mrs. Heidi Rattus Sample 4 Miss. Gertrude Musca Sample 5 Mr. Ivan Apis Sample 6 Blood on the envelope Sample 7 Hair found in Gertrude’s apartment. Assignment 2 Assignment 2 due Nov 10, 2008 Experimental set up 1. wild-type grown in the light 2. wild-type grown in the dark 3. det1 grown in the light 4. det1 grown in the dark -Extract RNA -RT mRNA -PCR cDNA Picture of Plants (WT and mutant) What are the expression levels of light regulated genes (chlorophyll a/b binding proteins CHAB and CLAB) using tubulin as loading control. CHAB CLAB CHAB or CLAB TUB Trizol Method • Why do we grind in liquid N2? • What does the Guanidium Thiocyanate do? • What does the Phenol do? • What does the Chloroform do? • What does the isopropanol do? Isolation of RNA using Commercial Trizol Reagent Trizol:-mono-phasic solution of phenol and guanidine isothiocyanate -lyses cells and dissolves cell components while maintaining integrity of RNA Liquid N2 Trizol Homogenize Phenol Aq Isopropyl alcohol Chloroform Org Ppt RNA 75% EtOH DEPC H2O RNA Chloroform: separate aqueous and organic phases (RNA is in the top aqueous phase) Isopropanol: Precipitates RNA 75% Ethanol: Wash RNA pellet DEPC (Diethylpyrocarbonate) water: Resuspend RNA pellet in RNAse-free water DEPC interacts with the active site histidine residue of RNAse and irreversibly inactivates RNAses. RNA preparation: Considerations • Yield: What controls yield? • RNases: What do we need to control this? • Quality of mRNA: What do we mean? • DNA contamination: Why is this important in RT-PCR? Dangers this week • TRIzol is very dangerous and can cause severe chemical burns. • Liquid N2 is very cold and can burn the skin; be careful and no fooling around. Original Northern blot Pepper et al., 1994 Cell 78, 109-116 RT-PCR: reverse transcriptase COMPONENTS TO ADD FOR REVERSE TRANSCRIPTION component 1 2 3 4 wild-type total RNA; light 5ml - - - det1 total RNA; light - 5ml - - wild-type total RNA ; dark - - 5ml - det1 total RNA; dark - - - 5ml water 5ml 5ml 5ml 5ml oligo dT primer 2ml 2ml 2ml 2ml Total volume 12ml 12ml 12ml 12ml COMPONENTS TO ADD FOR REVERSE TRANSCRIPTION Component Volume 5X Superscript buffer 4ml 0.1 DTT 2ml 10mM dNTP 1ml Final volume 20ml Add 0.5 μl Superscript reverse transcriptase. What do you think is in the RT buffer? Multiplex PCR • What is multiplex PCR? • What are the top two considerations when performing a multiplex PCR? What is in our multiplex PCR? • What is the control and what are light regulated? What is Multiplex PCR? (Combinatorial PCR) Amplifying various genes simultaneously (One template with multiple primer pairs) CHAB Multiplex PCR is used for: 1. 2. 3. 4. 5. DNA polymorphisms Mutations Detection of Pathogens Genetically modified organisms Quantitative RT-PCR CLAB CHAB or CLAB TUB Why are we amplifying (CHAB or CLAB) and tubulin simultaneously? RT-PCR • What is the problem with having DNA contamination? • Why is a set of exon primers that span an intron a good idea? Hint!!! What will be the outcome of an RT-PCR experiment if there is DNA contamination? Nice figure taken from Ann. Rev. Cell Dev. Biol 13, 203 Etiolated stage • Long hypocotyl (primary stem) • Apical hook • Unopened cotyledons (embryonic leaves) De-etiolated • • • • • Hypocotyl growth decreases Apical hook opens Cotyledons expand Chloroplasts develop A new gene expression program is induced Comparison of dark-grown (etiolated) and light-grown (de-etiolated) seedlings Etiolated characteristics De-etiolated characteristics No leaf growth Leaf growth promoted No chlorophyll Chlorophyll produced Rapid stem elongation Stem elongation suppressed Limited radial expansion of stem Radial expansion of stem Limited root elongation Root elongation promoted Limited production of lateral roots Lateral root development accelerated New Gene Expression Program is induced • Etiolation and de-etiolation are normal stages of plant development. • After germination there may be a lack of light and the seedling grows to find the light. Etiolated. • When sufficient light is found the seedling de-etiolates a developmental process that optimizes the body plan for photosynthesis. Picture of DET1 function DET1 is a nuclear protein with a nuclear localization sequence. DET1 is part of a protein complex with COP10 and DDB1 called CDD CDD stimulates ubiquitin-conjugating enzymes. Ubiquitination of HY5 (required for normal light development) -targets it for degradation. DET1 function in light and dark Why have det1 mutants lost this response? No DET1; HY5 is not degraded and can not initiate etiolated growth. Det1 in Humans? • Human de-etiolated-1 regulates c-Jun by assembling a CUL4A ubiquitin ligase. Science 303, 1371-1374 (2004). • Jun is an oncogene! Discussion of Article 4