A Novel Approach to Identifying Differential Gene Expression Alexander Richardson Robert Pignolo

Methods used to identify differential gene expression Subtractive cDNA libraries and probes Differential display Microarray analysis 2-D protein electrophoresis

2-D separation of RNA by length and secondary structure First dimension: separation by length (molecular weight) using glyoxal denaturation Second dimension: separation by secondary structure after renaturation (occurs at pH>8 with ammonium hydroxide)

Glyoxal Denaturation of RNA Binds to guanosine preferentially, but can bind to all bases at high concentrations The glyoxal-guanosine adduct is readily reversible at ph >8 using ammonium hydroxide

2-D Separation of RNA Length (molecular weight) Secondary structure

2-D Separation of RNA: 1 st /2 nd Dimension Length (molecular weight) 28S 18S Length (molecular weight) Secondary Structure

Identification of separated RNA I Excise band Isolate RNA Perform cDNA synthesis Clone into sequencing vector Submit for sequencing

Identification of separated RNA II Migration pattern in two dimensions should permit mapping based on specific coordinates Coordinates can be measured relative to most abundant RNAs (e.g., 28S and 18S rRNAs)

The First 30 Tries Optimization –Stain – Acridine Orange, Ethidium Bromide, GelStar Stain (pre vs. post staining) –Large Format vs. Small Format Gel –pH treatment – chemical (NH4OH,NaOH), concentration, pH, duration –RNA type – wholesale, mRNA, polyA, marker

Recent Results Optimization –Large Format Gel –RNA marker ( kb, 9 bands) –GelStar Nucleic Acid Stain (pre-stain) –NH4OH Treatment (2 M) for 1-15 mins. –Glyoxal pH >5 (prepared fresh, avoid oxidation)

One Dimension - glyoxal + glyoxal

Attempts at Glyoxal Removal +g No treatment 3 M 1 M

Successful Glyoxal Removal All lanes glyoxalated Lanes 2-5 treated with 2M NH4OH No treatment 1 min. 5 min.10 min.15 min.

Repeat Exp. Using 1 M NH4OH All lanes glyoxalated Lanes 2-5 treated with 2M NH4OH No treatment1 min.5 min.10 min.15 min.