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 Length (molecular weight) Secondary structure

2-D Separation of RNA: 1 st Dimension Length (molecular weight) 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 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)

Attempt #1… Length (molecular weight) 28S 18S

Testing Acridine Orange Re-done using mini-gel and non-denatured RNA Expected results: red  single-stranded green  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 + EtBr Lane 2  RNA sample + EtBr Lane 9  DNA sample + Acridine Orange Lane 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