a novel approach to identifying differential gene expression alexander richardson robert pignolo

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

Leng

th (

mol

ecul

ar

wei

ght)

Secondary structure

2-D Separation of RNA: 1st/2nd

DimensionLe

ngth

(m

olec

ular

w

eigh

t)

28S

18S

Length (molecular weight)

Sec

onda

ry S

truc

ture

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 (0.2-10 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 + glyoxal

Attempts at Glyoxal Removal

+g +g+g

No treatment 3 M 1 M

Successful Glyoxal Removal

All lanes glyoxalated

Lanes 2-5 treatedwith 2M NH4OH

No treatment 1 min.1 min. 5 min. 10 min. 15 min.

Repeat Exp. Using 1 M NH4OH

All lanes glyoxalated

Lanes 2-5 treatedwith 2M NH4OH

No treatment 1 min. 5 min. 10 min. 15 min.