A Novel Approach to Identifying Differential Gene Expression Robert J. Pignolo and Oriana...
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Transcript of A Novel Approach to Identifying Differential Gene Expression Robert J. Pignolo and Oriana...
A Novel Approach to Identifying Differential Gene Expression
Robert J. Pignolo and Oriana Galardi-Este
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)
2-D Separation of RNA
Leng
th (
mol
ecul
ar
wei
ght)
Secondary structure
2-D Separation of RNA: 1st Dimension
Leng
th (
mol
ecul
ar
wei
ght)
28S
18S
Non-radioactive detection of RNA
UV light alone (limit = 4.2 ug) Methylene blue (limit = 2.1 ug) Ethidium bromide (limit = < 0.5
ug) Acridine orange (0.05 ug dsRNA;
0.1 ug ssRNA)
Detection of RNA using acridine orange
Leng
th (
mol
ecul
ar
wei
ght)
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)
Attempt #1…Le
ngth
(m
olec
ular
w
eigh
t)
28S
18S
Testing Acridine Orange
Re-done using mini-gel and non-denatured RNA
Expected results: red single-strandedgreen double-stranded
So mostly green because no glyoxal used
Actual Result
Attempt # 2…
Lane 1: Denatured Sample (Treated with glyoxal)Lane 2: Non-denatured Sample (Not treated with glyoxal)
Results…
Questions to Answer…
Why am I only seeing 28S and 18S big bands? Where are all the smaller bands?
What kind of UV illuminator will let me see the red and green expected results from the acridine orange?
Why are bands in Acridine Orange test smeared? Why are the bands all red if they are not denatured?
Next Step:
Run mini-gel with:Lane 1 DNA sample + EtBrLane 2 RNA sample + EtBrLane 9 DNA sample + Acridine OrangeLane 10 RNA sample + Acridine
Orange
Result
Next Try:
Newly found paper mentions staining and de-staining of Acridine Orange should be done in the dark
SO, we repeated experiment without EtBr and with staining done in dark and we found…
Results
DNA RNA
After 1 hour of de-staining…
DNA RNA
After 4 days of de-staining…
TOTAL FLUKE
Yay! Now what?
Now we’ve seen the green and red. We know the UV light box works. Now we need to test whether
denatured and non-denatured RNA are distinguishable from each other in terms of color and migration
Results
2 clear green DNA bands
2 clear red non-denatured RNA bands
Non-denatured RNA migrated further than denatured RNA (expected)
Denatured RNA very fuzzy and possibly more red?
DNA RNA DENATURED
What about the big-Gels?
All the while, nothing is working on the big-gels…gels keep turning out clear
Explanation: Glyoxal Loading Buffer made with wrong concentrations!
Results after changing buffer
Finally saw 2 red bands…
But where are all the other bands??
Next 3 experiments:
1) DNA / nRNA / gRNA run on mini-gel, stained with EtBr + AO
2) DNA / nRNA / gRNA run on mini-gel stained with AO only
3) DNA / nRNA / gRNA run on 1.5% mini-gel stained with AO only
In all 3, nRNA and gRNA treated identically
Result 1
Result 2
Result 3
So Far…
Why am I only seeing 28S and 18S big bands? Where are all the smaller bands?
Still don’t know… What kind of UV illuminator will let me see the red
and green expected results from the acridine orange?
Turns out it was a de-staining problem Why are bands in Acridine Orange test smeared?
Why are the bands all red if they are not denatured? Still having problems with this – unsure why bands
are not as sharp as they are when stained with EtBr