Introduction to Neurospora Microarray Methods

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http//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
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Neurospora 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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ID 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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384 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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ArrayControl 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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Neurospora 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.
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Etch the four corners of microarray spots onto
the back with a diamond pen. ? Proceed to
Rehydration and UV-cross link.
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Rehydrate 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
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Spots before hydration after hydration
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UV 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)
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RNA 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
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cDNA synthesis home-assembled chemicals (2040
/ hyb) Or Kits Promega/Corning (80 / hyb)
includes hyb reagents Invitrogen (30 /
hyb) Amersham (35 / hyb)
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cDNA 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
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mRNA
ArrayOligoSelector By Jing Zhu, UCSF
AAAA 3
UTR
Start
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Stop
oligomer
Median 764 bp
Mean 1066 bp
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hyb36
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)
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rRNA background or oligos distant from 3-end
hyb33
Total RNA oligo dT N6
Total RNA oligo dT
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Oligo 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
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Load sample to pre-soaked / pre-hybridized array
? Hybridization at 42C for overnight ?
wash ?
Axon Scanner Face Down, Barcode to your side
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Acknowledgements 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