Supporting Information Methods S1 Supplemental Materials and Methods
MiRNA microarray
MiRNA samples isolated from the young leaves and roots of control or stress-treated
S. bicolor plants were used for miRNA microarray hybridization. All samples were
assessed for the presence of enriched miRNA using an Experion Bioanalyzer
(Bio-Rad, Hercules, CA, USA). The plant GeneChip® miRNA 3.0 Array (Affymetrix,
Santa Clara, CA, USA) was used in this study, and contained 3741 well-characterized
plant miRNAs from Sorghum bicolor, Arabidopsis thaliana, Zea mays, Oryza sativa,
Glycine max, and other species as noted in the miRBase v17 coverage and various
controls (http://www.mirbase.org/). The miRNAs were labeled using the FlashTag
Biotin RNA labeling kit (Genisphere, Hatfield, PA, USA) according to the provided
protocol and then hybridized to Affymetrix GeneChip miRNA 3.0 microarrays. Three
replicates each for leaf and for root, using a total of 12 arrays, were applied to
compare the control and Zn deficiency conditions. The Affymetrix GeneChip 3000
TG microarray hybridization was performed with the assistance of Biochip
Corporation (Tianjin, China). Array hybridization, washing, and staining were
performed per the manufacturer's instructions, and arrays were scanned with a
GeneChip Scanner 7G 4C. Only plant miRNAs were considered in our analysis. The
Affymetrix miRNA QC Tool software (Affymetrix, Inc.) was used for data
summarization, normalization, and quality control. The miRNAs with P < 0.05 and
fold change > 2.0 compared to the control were defined as differentially expressed.
5’ RACE of miRNA cleavage
For mapping the internal cleavage site in the predicted target genes of the miRNAs,
RLM-RACE was performed using the GeneRacer Kit (Invitrogen, USA). A modified
procedure for RLM-RACE was performed following the GeneRacer Kit instructions
as described previously (Song et al. 2009). Total RNA was extracted from the leaves
and roots of S. bicolor seedlings using TRIzol reagent (Invitrogen, USA). The RNA
quality and integrity were checked before the cDNA was synthesized using a Bio-Rad
Experion RNA StdSens analysis kit (Bio-Rad, Hercules, CA, USA). Poly(A)+ mRNA
was purified from the pooled RNA from all tissues using a PolyA kit (Promega,
Madison, WI, USA) according to the manufacturer’s instructions. The GeneRacer
RNA Oligo adapter was directly ligated to mRNA without calf intestinal phosphatase
and tobacco acid pyrophosphatase treatment. The GeneRacer Oligo dT primer was
then used to synthesize first-strand cDNA. This cDNA was subjected to an
amplification procedure with the GeneRacer 5’ Primer and the GeneRacer 3’ Primer
to generate a pool of non-gene-specific 5’RACE products. Gene-specific 5’RACE
reactions were performed with the GeneRacer 5’Nested Primer and gene-specific
primers (Table S1). In each case, a unique gene-specific DNA fragment was amplified.
After amplification, 5’RACE products were gel-purified and cloned into the pMD
19-T vector (TaKaRa, Otsu, Japan), and at least 10 independent clones were randomly
chosen and sequenced.
RT-qPCR analysis of gene expression
The RNA was extracted from the young leaves, mature leaves, and roots of control or
stress-treated S. bicolor plants using TRIzol (Gibco/BRL, Life Technologies). The
RNA quality and integrity were checked before the cDNA was synthesized using the
Bio-Rad Experion RNA StdSens analysis kit (Bio-Rad, Hercules, CA, USA). For the
quantitative RT-PCR (RT-qPCR), we first accurately quantified the RNA
concentration using spectrophotometry. The cDNA was synthesized from the
DNase-treated total RNA (1 µg) using a Reverse Transcription System Kit (Promega)
and oligo (dT) primers. The cDNA produced was diluted 1:15, and 3 μl was used in
the RT-qPCR reaction in a 7500 Real-Time System (Applied Biosystems) using
Platinum® SYBR® Green qPCR SuperMix-UDG (Invitrogen, USA). Based on
semi-quantitative RT-PCR analysis and the Genevestigators’ RefGenes tool V3 (Hruz
et al., 2008), we selected ACT (Sb01g003250), GADPH (Sb04g025120), UBI3
(Sb01g030340), UBQ (Sb04g031060), TuA2 (Sb01g009570), and EF-1alpha
(Sb02g036420) as the candidate reference genes for RT-qPCR normalization. The
geNorm software (http://medgen.ugent.be/~jvdesomp/genorm/) was used to choose
the most suitable reference genes. The qRT-PCR for each gene was done on three
biological replicates with duplicates for each biological replicate. The relative
transcript level was determined for each sample and averaged over the six replicates.
Specific primers for each gene are listed in Table S1. All primer pairs produced only
one peak in DNA melting curves indicating high specificity of the primers.
To verify the gene expression patterns of target genes, the RT-qPCR analysis was
performed for selected genes. We selected ACT, GADPH, UBI3, UBQ, TuA2, and
EF-1alpha as the candidate reference genes for RT-qPCR normalization. The
expression of ACT was the most stable in Sorghum, and the pairwise variation V value
of ACT and TuA2 was 0.11 (Fig. S1), which suggested that the two genes were
suitable reference genes for RT-qPCR normalization.