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