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

Today’s Laboratory Objectives 1. To isolate high quality total RNA from E. huxleyi cells grown in nutrient rich (F/2) versus phosphate limiting (F/2) media 1. To quantitate the amount and purity of the total RNA isolated 2. To become familiar with the nuances in handling RNA

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

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

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 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= is optimal for RNA

Next Week RNA Electrophoresis cDNA Synthesis