Quantitative Real-Time PCR (qRT-PCR) validation of ATH1 arrays
A set of 23 DEGs from microarray analysis of wus mutants were selected for validation by qRT-PCR.
Primers were designed using Quantprime (Arvidsson et al 2008) to amplify 175-250bp fragments
spanning introns (Table S.I. 1 below). Total RNA was isolated using the RNA Easy kit (Qiagen) and treated
with DNase I (MBI Fermentas). First strand synthesis was performed using First Strand cDNA synthesis
kit (MBI Fermentas). The qRT-PCR was performed using CFX96 Real-Time PCR detection system (Bio-Rad
Laboratories) with iQ SYBR Green supermix (Bio-Rad Laboratories) and analyzed with CFX Manager
software (Bio-Rad Laboratories). Reactions were carried out in triplicate. Melt-curve analysis was
performed to confirm specificity of the amplification reaction and normalization was performed with
primers specific for eukaryotic initiation factor 1a (At5g60390) and actin-2 (At3g18780). The majority of
DEGs showed similar differential expression in both microarray and qRT-PCR analysis (Figure S.I. 1
below).
Gene Id
AT1G15040
AT1G15040
AT5G19170
AT5G19170
AT2G20180
AT2G20180
AT1G11350
AT1G11350
AT5G39520
AT5G39520
AT1G62320
AT1G62320
AT4G21200
AT4G21200
AT5G55470
AT5G55470
AT5G04230
AT5G04230
AT2G05160
AT2G05160
EF1a
EF1a
ACTIN
ACTIN
At1g15040
At1g15040
At1g51960
At1g51960
At3g26900
At3g26980
At2g15790
At2g15790
At2g37860
At2g37860
At1g77740
At1g77740
At4g30950
At4g30950
At1g04230
At1g04230
At3g08010
AT3G08010
At5g52330
At5g52330
At5g53810
At5g53810
Primer
Forward/Reverse
(F/R)
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
R
F
F
R
F
R
F
R
Sequence
GGC TGC GCT TTA GTT TAT CAG GAG
AGC CTC TAG AAA CTC AGC TTG GG
TCG CTC AGA TTT GGC TGG CAT C
CCA TCC GCC GTC CAA TAT GTA AAC
ACT TAT ACC TCG CTG CAA CAA GTC
GTT GCA TGC CCG GAT ACA TCA TTG
CAA AGC TGC AAC ACC GGA ATC TC
TCT TTG CTT CAC CGG GTC AAA TAG
TGA CGA AAT GGG AGC TTC AAG GG
TCT GGT ACA AGC GCG AGA ACT G
AGC TAT GGA TCC GGG ACA GAT TG
ACC CGC ACT CTC ATA CTT CTG ATG
TGG TGA CTT ATT TCA GGC ATG GAG
TAA CGG CGT CGT ATG ATG GAC AC
ACG AGC AGC ATT TGT CTT TCC AC
AAC ACC GGA GTA TGT GAA CTG C
TTG ATG ACC CAA GCA GCC TCA C
AAT TTC AGC TCG GCC TCG AAC G
TTA CTC CTA CCT CGG CTT CTC C
CAA ATG CGG AGC AAG AGG AAC A
TGA GCA CGC TCT TCT TGC TTT CA
GGT GGT GGC ATC CAT CTT GTT
GGT AAC ATT GTG CTC AGT GGT GG
GAC CTG CCT CAT CAT ACT CG
GGA AGA CCA CCA CAC TCG TC
GTG GAA GGA AAG CAT ATC AGA CAG C
CAT CAC GAA GGA GGA GCG G
GTT GTA CTC TTT GTT GAG GCT GCG
TTC ATG ACT GTT GCA TCT CTC AAG G
GCA GCA TAC ACA GTG ACT CTT CC
CCC CAC CTT GTA TCA TAA ACC CC
CTC TTT TGA ACT TCA GCA ACA GTG GC
GAA GCT GCT AAG AAC TAT GGG ATC CG
CTC TTT TGA ACT TCA GCA ACA GTG GC
CTT GTG GAC GTC GCA GTG AGA G
CTG TTG CAA CAA AGT GTC AAA ACT AG
GCT TCT TCT GCT CGT GTT TCT CC
TTC TGC ACT GTC AGC TGA AGG
CGG AAA TGG ATA TCT TGC TGC TTC
GGA TTG CGC GTG GAG AAA AAA CC
CAT TGT TAC CAT CAC TCA AGA CTT GG
CAC AGT GTC ATA ACG TTC TTG CAA CC
GTGGACCGACCTTTCTTCCTTG
CACCGAAGCTTGTCCATTTGGC
GAGGACTTGGCAACACACTAGGTC
CCACATGGTTCACACCGGGATAAG
Table S.I. 1. Primers used for qRT-PCR validation of array data.
Figure S.I.1. A set of 23 genes showing statistically significantly increased or decreased expression
between wus GABI-KAT mutant (GABI_870H12) and wild type in the ATH1 arrays were selected for
validation by qRT-PCR. The graph represents a scatter plot of Log 2 ratios of expression in the wus
mutant and wild type after 30 hrs treatment with the cytokinin 2iP. qRT-PCR values (X axis) and ATH1
array values (Y-axis) are plotted against each other. Most genes show a strong correlation between the
two data sets, either in up-regulation (upper right quadrant) or down-regulation (lower left quadrant).
Characterizing wus mutants.
The RNA product produced by the SAIL and GABI-KAT alleles of WUS was examined using semiquantitative RT-PCR. Primers 63F(CATCACGGTGTTCCCATGC) and 23R(GACCAAAC-AGAGGCTTTGCT)
spanning the insertion site amplified a 310bp product from wild type CDNA after 30 hours of 2iP
induction, when WUS transcriptional reporters are expressed within transdifferentiating LRP. Whereas
no product was amplified from wild type samples at the 0 hour time point (when no WUS reporter
expression is observed), or from homozygous mutants for either of the wus alleles at both time points.
Conversely primers Wus 11F (CAAGCTCAGGTACTGAATGTGG) and Wus7R (CCATACTTCCAGATGGCAC),
which were both located downstream of the insertion sites, amplified a 320bp product in both wus
mutants after 0 and 30 hours 2iP. Whereas the same primer combination only yielded this product in
the 30 hour 2iP sample of wild type and not the 0 hour sample. Primers for housekeeping genes UBC
(F:TCAAATGGACCGCTCTTATC, R:CACAGACTGAAGCGTCCAAG) and Ran1(F:GTCATTGTTGGTGATGGAGGC,
R: CATTCTTGTATGTGAGCCGTGC) were used as controls. Fully quantitative PCR showed a 50 fold higher
amplification for the same primers in the mutants compared to wild type. These results suggest that
there is increased expression of a truncated RNA product in the wus mutants even in the absence of a
cytokinin treatment necessary for expression of WUS transcriptional markers in the root of wild type
plants. Similar mis-expression of RNA products adjacent to insert sites, driven by TDNA promoter
sequences, has been previously reported (Ulker et al. 2008).
To understand the T-DNA insertion features of the GABI-KAT wus mutant (GABI_870H12) a left border
(LB)
primer
(ATATTGACCATCATACTCATTGC)
and
GABI-KAT
Right
border
(RB)
primer
(GTGGATTGATGTGATATCTCC,) were used to amplify flanking sequences from GABI-KAT mutant. The
right and left primer (RP: TAGATGCAAATTTGGTTTGCC & LP: AAAACCTTCTTTTTGGGTCCC) flanking the
TDNA insert as given by the GABI-KAT database was used. While LB and RP primer amplified a product of
expected size, RB and LP gave none suggesting aberrant TDNA insertion. When the LB was used with LP
an unexpected product of 750bp size was amplified in the GABI-KAT mutant and not in the controls. This
suggests an inverted duplication at the insertion location, with cis-elements within the insert driving
transcription of a truncated wus product.
Ulker, B., Peiter, E., Dixon, D.P., Moffat, C., Capper, R., Bouche´, N., Edwards, R., Sanders, D., Knight,
H. and Knight, M.R. (2008) Getting the most out of publicly available T-DNA insertion lines.
Plant Journal, 56, 665-677.