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Gene Expression

Gene Expression. RNA Isolation from Emiliania huxleyi. Broad and Long Term Objective. To characterize the expression of metacaspase in E. huxleyi cells grown in phosphate limiting media (F/50) versus nutrient replete media (F/2). Research Plan.

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Gene Expression

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  1. Gene Expression RNA Isolation from Emiliania huxleyi

  2. Broad and Long Term Objective To characterize the expression of metacaspase in E. huxleyi cells grown in phosphate limiting media (F/50) versus nutrient replete media (F/2)

  3. Research Plan RNA Isolation from cells grown in F/2 and F/50 media RNA Electrophoresis Assessing Gene Expression Northern Blot RNase Protection Quantitative PCR

  4. Today’s Laboratory Objectives • To isolate high quality total RNA from E. huxleyi cells grown in nutrient rich (F/2) versus phosphate limiting (F/2) media • To quantitate the amount and purity of the total RNA isolated • To become familiar with the nuances in handling RNA

  5. CAUTION: RNases ARE EVERYWHERE! Control of RNases • Wear Gloves and Practice Sterile Technique • Use Disposable Plastics or Baked Glassware • Use chemicals or reagents that will inactive RNAses (DEPC treated water, chaotropic agents, etc) • Always Keep RNA on Ice or Frozen • Work quickly and carefully

  6. Guanidinium Thiocyanate Extraction • Cells harvested, resuspended in ETOH and frozen • Cell lysis accomplished via grinding in liquid nitrogen • Guanidinium thiocyanate= chaotropic agent inhibits RNases • Sarkosyl= disrupts membranes and inhibits ribonucelases • B-mercaptoethanol= reduces disulfide bonds of RNAses and prevents free-radical crosslinking of phenolics to DNA

  7. Theoretical Basis of UV Spectrophotometry for Quantitating Amount and Purity of RNA To Quantify your RNA sample: A260 x Dilution Factor x 40 ug/ml= concentration of nucleic acids in a sample using a 1 cm pathlength To estimate the purity of your sample: A260/A280= ratio of nucleic acids/protein A260/A280= 1.8-2.1 is optimal for RNA

  8. Next Week RNA Electrophoresis cDNA Synthesis

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