Gene Expression

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
Todays 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