1. RNA Electrophoresis

2. Broad and Long Term Objective To characterize the expression of metacaspase in E. huxleyi cells grown in phosphate limiting media versus nutrient rich media To characterize the expression of metacaspase in E. huxleyi cells grown in phosphate limiting media versus nutrient rich media

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 1. To assess the integrity of the RNA e extracted last week using agarose gel electrophoresis. electrophoresis. 2. To make cDNA from total RNA using Reverse Transcriptase and an oligo dT primer 3. To assess the success of the cDNA synthesis reaction and devise a strategy using real time RT PCR to examine the expression of metacaspase in cells grown in phosphate limiting and phosphate replete media

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. RNA Electrophoresis RNA is highly susceptible to intrastrand H-bonding; such secondary structure will affects its migration through an agarose gel unless it is resolved Denaturing Agarose Gel Electrophoresis  Two types: 1) Formaldehyde 2) glyoxyl dimethylsulfoxide  Formamide and formalydehyde are included in Loading Buffer  RNA samples heated at 65° C for 5 minutes prior to electrophoresis

7. Expected Results  Two prominent rRNA Bands  Slight smear of various sized mRNA molecules in background

8. Ribonuclease Protection Assay (www.ambion.com)

9. Comparative (Quantitative) RT- PCR (www.ambion.com)

10. Northern Blot

11. Real Time RT PCR to Validate Differentially Expressed Genes Reveal by SSH Sample---RNA Isolation---cDNA Synthesis---RT PCR Amplification Real Time PCR Work Flow

12. Defining Parameters of Real Time RT PCR Cycle Threshold: Cycle # when product fluoresence exceeds that of background Fold Change: 2ΔCt Melt Curve: fluorescence plotted as a function of temperature as thermal cycler heats through dissociate temperature of product