Title: Introduction to Neurospora Microarray Methods
1http//web.uconn.edu/townsend/Links/ffdatabase/dow
nloads.html
Introduction to Neurospora Microarray Methods
Takao Kasuga, Univ. California, Berkeley Brief
description of Neurospora microarrays After
acquisition from FGSC Rehydration UV-Cross
link RNA isolation and cDNA synthesis Hybridiza
tion
S. Brown B. Gilbert
L. Glass J. Taylor CG Tian
2Neurospora Microarrays 70-mer oligos designed
based on 10,482 ORFs MIPS 9,351 Broad
Institute 1,128 rRNA genes 3 ChIP-chip
oligos 384
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3ID Name 3nc442_550_1875 NCU00340.1 Distance
from stop codon In 160 cases, multiple MIPS
ORFs in one large NCU ORF ID Name 1nc310_320_71
1 NCU01921.1a 1nc310_330_1762 NCU01921.1b 1nc310
_340_611 NCU01921.1c
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4384 oligos designed for Chromatin
Immunoprecipitation-chip Names are all
ChIP-chip ID Intergenic NCU02431.M__NCU02432.
M__2_258 Genic NCU02432.1__1_423 Genic reverse
complement NCU02432.1__3_267_RevCom
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5ArrayControl Sense Oligo Spots 18 (Ambion)
for ArrayControl RNA Spike 18
(polyadenylated bacterial mRNA)
ID ID Spot1 Spot1_half Spot2 Spot2_half Spot3
Spot3_half Spot4 Spot4_half Spot5 Spot5_half Spo
t6 Spot6_half Spot7 Spot7_half Spot8 Spot8_half
400 pmol 200 pmol
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6Neurospora Microarrays from FGSC ? First, find a
production date of arrays ? Go the Nc Functional
Genomics Database website http//web.uconn.edu/tow
nsend/Links/ffdatabase/downloads.html ? Open
N. crassa GAL ReadMe and download an appropriate
array grid (gal) file. GeneTemplate.xls, gene
name, ID, annotation, FunCat, oligo sequences.
7Etch the four corners of microarray spots onto
the back with a diamond pen. ? Proceed to
Rehydration and UV-cross link.
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8Rehydrate to evenly distribute DNA within each
spot ? On 80c heat block
Slides suspended over 1x SSC in a humidity chamber
dried array 1 min 7 mins
9Spots before hydration after hydration
10UV cross link at 600mJ ? Store in desiccator at
RT (gt1 year) ? w/i 3 days prior to
hybridization, Presoak slides with NaBH4
(Pronto Background Reduction Kit) (175 for 50
slides) ? 2 hours prior to hybridization, Start
Prehybridization (The UNM BSA protocol or
Promega/Corning kit)
11RNA isolation
100mg wet-mycelium or 6 cm2 mycelium grown on
cellophane ? Trizol and beadbeater ? lt 500 µg
total RNA ? RNeasy mini column (Qiagen), DNase
(optional) if cellophane is used ? lt 100 µg
12cDNA synthesis home-assembled chemicals (2040
/ hyb) Or Kits Promega/Corning (80 / hyb)
includes hyb reagents Invitrogen (30 /
hyb) Amersham (35 / hyb)
13cDNA synthesis
Total RNA or mRNA spike ArrayControl mRNA
(Ambion) oligo dT or oligo dT Random
hexamer ? Reverse Transcription with
aminoarryl-dUTP ? Conjugation of Cy dyes to
aminoarryl cDNA ? Spec, Quantification /
incorporation efficiency (NanoDrop
ND-1000) ? Hybridization
14mRNA
ArrayOligoSelector By Jing Zhu, UCSF
AAAA 3
UTR
Start
5
Stop
oligomer
Median 764 bp
Mean 1066 bp
15hyb36
Cy3 total RNA oligo dT
Cy5 total RNA oligo dT
20 ?g of total RNA or 2 ?g of mRNA (at least 250
?g of total RNA)
16rRNA background or oligos distant from 3-end
hyb33
Total RNA oligo dT N6
Total RNA oligo dT
17Oligo dT
Oligo dT dN6
High Signal /High Noise
Ok Signal/Ok Noise
Total RNA
Ok Signal/Ok Noise
High Signal/Low Noise
mRNA
cDNA synthesis
18Load sample to pre-soaked / pre-hybridized array
? Hybridization at 42C for overnight ?
wash ?
Axon Scanner Face Down, Barcode to your side
19Acknowledgements Gertrud Mannhaupt (MIPS)
Audrey Gasch (UW-Madison) Jing Zhu, Adam Carol,
Joe DeRisi (UCSF) James Galagan (Broad
Institute) Patty Holman (UCB) Mike Freitag, Eric
Selker (Oregon) Support from NIH