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Chapter 8: Manipulating DNA, RNA and proteins Isolating the correct tissue Analysing DNA in tissues Accuracy with numbers • Blond hair Accuracy with numbers • Blond hair • Female Accuracy with numbers • Blond hair • Female • Blue pants Accuracy with numbers • • • • Blond hair Female Blue pants Black shoes Accuracy with numbers • • • • • Blond hair Female Blue pants Black shoes Pony tail Accuracy with numbers • • • • • • Blond hair Female Blue pants Black shoes Pony tail White shirt Accuracy with numbers • • • • • • • Blond hair Female Blue pants Black shoes Pony tail White shirt Short sleeves Accuracy with numbers • • • • • • • Blond hair Female Blue pants Black shoes Pony tail White shirt Short sleeves • Glasses Accuracy with numbers • • • • • • • Blond hair Female Blue pants Black shoes Pony tail White shirt Short sleeves • Glasses • Under 5’5” Measuring and changing gene expression How much RNA is there? RNA quantification How can the levels of RNA be altered? Increasing and decreasing RNA levels Assessing rates of transcription Reporter genes Quantifying RNA Methods are available to measure RNA levels: -within the cell (in situ hybridization) In situ hybridization of fluorescent cDNA to bind to mRNA (presence or absence of expression) Quantifying RNA Methods are available to measure RNA levels: -within the cell (in situ hybridization) -in tissue samples (northerns, DNA arrays) Tissues must be broken and the integrity of RNA preserved by inhibiting RNA degradation by RNases RNA is purified (several ways) and quantified by: -northern hybridization -reverse transcription -PCR (RT-PCR) -cDNA arrays Quantifying RNA Northern blots: electrophoresis to separate RNA, transfer to membranes, hybridize with a single radioactive cDNA Quantifying RNA RT-PCR: ultrasensitive way to measure levels of specific RNAs 1 2 5 10 20 50pg Product 2ul Real-time PCR: very rapid (and sensitive) way to measure mRNA by combining fluorescent detection with PCR 10 pg RNA in the 2ul unknown # of PCR cycles Quantifying RNA cDNA arrays: compare 1000’s of RNA’s in treatment pairs (ie control vs treated) Bioinformaticists help interpret vast amounts of array data 1 2 -genes are grouped into characteristic patterns 0 30 1h 2h 4h 8h 12h 24h 3 4 Proteins Altering gene expression (1) Prevent protein from working -add inhibitors -microinject antibodies -dominant negative transgenes Altering gene expression (2) Reducing RNA levels by enhancing RNA degradation or blocking translation -anti-sense (injections of RNA, oligonucleotides or morpholinos) -RNA interference (RNAi) Altering gene expression (3) Alter genes -random mutagenesis -targeted knock-outs -gene replacement or addition Transfecting cells, including bacteria Living bacteria (and other cells) can be transfected with DNA Cells can be treated to enable them to take up DNA (competent cells) Cells can be permeabilized by high voltage (electroporated) to take up DNA DNA can be packaged in liposomes that get incorporated into membranes DNA can be packaged into viruses that infect cells