BE115+-+Lab08a - Lab 8a: RNA purification and...

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Page 1 of 6 Bioengineering 115: Cell Biology for Engineers Laboratory 8a: RNA Purification and Quantification Last updated: January 21, 2011 Lab 8a: RNA purification and quantification 1 Introduction Thus far you have learned how to culture mammalian cells and how to analyze the growth of these cultures by cell counting, protein assay, SDS-PAGE, and Western blotting. All of these methods measure what is present in the culture at a given moment. The next series of techniques that you will master is the analysis of what the cells in a culture are producing. As you know, proteins are produced following a series of events that include transcription and translation of genes. Genes are said to be expressed when the DNA for that gene is transcribed into messenger RNA (mRNA). In most (though not all) instances high mRNA copy number results in a high amount of protein production (translation). The half-life of mRNA in the cell is typically very brief (60 minutes). Therefore measurement of a specific mRNA tells us if the cells of a culture (or tissue) are expressing a particular gene at the time we extract the RNA. There are many methods for extraction of RNA. Common steps include cell lysis, denaturation of enzymes that degrade RNA (called RNases), and selective precipitation or purification of total or mRNA.
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Page 2 of 6 Bioengineering 115: Cell Biology for Engineers Laboratory 8a: RNA Purification and Quantification Last updated: January 21, 2011 2 Objectives To extract mRNA from cultured cells To measure RNA concentration of your extracts To prepare RNA for RT-PCR 3 References and Links RNeasy kit manual (http://www.qiagen.com/literature/render.aspx?id=352) 4 Reagents, Supplies, and Equipment 4.1 Reagents 1. Buffer RLT (for lysis) 2. RNase-free 70% ethanol 3. Buffer RW1 (first wash) 4. Buffer RPE (second wash) 5. RNase-free water 6. RNase-free TE buffer 7. PBS 4.2 Supplies 1. RNeasy kit (Qiagen 74104) 2. Filter-tip pipette tips 3.
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BE115+-+Lab08a - Lab 8a: RNA purification and...

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