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Plant Molecular Biology Reporter 22: 325-337, December 2004 9 2004 International Society for Plant Molecular Biology. Printed in Canada.
Commentary
Comparison of Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Analysis of Gene Expression in Sugarcane HAYATI M. I S K A N D A R m'3'4'*, R O B E R T S. S I M P S O N l, R O S A N N E E. C A S U 2'3, G R A H A M D. B O N N E T T 2'3, D O N A L D J. M A C L E A N 1 and J O H N M. M A N N E R S 2'3
1School of Molecular and Microbial Sciences, University of Queensland, St Lucia, Queensland 4067, Australia; 2CSIRO, Plant Industry, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, Queensland 4067, Australia; 3Cooperative Research Centre for Sugar Industry Innovation through Biotechnology, University of Queensland, St Lucia, Queensland 4072, Australia; 4Indonesian Biotechnology Research Institute for Estate Crops, J1. Taman Kencana, No. 1, Bogor 16151, Indonesia Abstract. A protocol for reverse transcription followed by real-time quantitative PCR (RTqPCR) analysis of tissue-specific and genotype-variable gene expression in sugarcane (Saccharum sp.) was developed. A key requirement for this analysis was the identification of a housekeeping gene with transcript levels that were relatively stable across tissues and genotypes, suitable for use as a reference. Primers for ~-actin, ~-tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes and 25S ribosomal RNA were designed and tested by RT-qPCR, and formation of product in the reactions was measured with the SYBR green I dye system. Ribosomal RNA was the most sensitive and consistent as a reference gene. Determination of the expression levels of ~-actin, ~-tubulin, and GAPDH transcripts relative to that of 25S rRNA showed that GAPDH had the most consistent mRNA expression of protein-coding genes across different tissues. GAPDH also showed low variation in expression in maturing stem internodes when compared across 2 cultivars and 3 other Saccharum species. GAPDH therefore appears to be a suitable "housekeeping gene" in addition to 25S rRNA as a reference for measuring the relative expression of other genes in sugarcane. With use of GAPDH as a reference, the relative expression of the sugarcane sugar transporter gene Pst2a was assessed in a range of tissues. The result obtained was similar to our previously published Northern blot analysis. The protocol described here, using GAPDH as a reference gene, is recommended for studying the expression of other genes of interest in diverse tissues and genotypes of sugarcane.
Key words: gene expression, reference genes, RT-qPCR, sugarcane (Saccharum) Abbreviations: DEPC, diethyl pyrocarbonate; EST, expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase, rRNA, ribosomal RNA; RT-qPCR, reverse transcription followed by quantitative real-time PCR.
*Author for correspondence, e-mail:
[email protected]; fax: +61-7-3214 2950; ph: +61-7-3214 2334.
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Introduction The ability of sugarcane to accumulate high concentrations of sucrose makes it a valuable crop in the tropics and subtropics. The identification of genes that control and facilitate the accumulation of sucrose in the sugarcane stem would provide useful tools for increasing the concentration of sugar in sugarcane, by means of either improved selection or genetic modification. Recently, many genes expressed in sugarcane stems and other tissues have been detected by analysis of expressed sequence tags (ESTs) (Carson and Botha, 2000; Vettore et al., 2001+Casu et al., 2003). The differential expression of a large number of genes can simultaneously be analyzed by macro- or microarrays (Casu et al~.r 2003, 2004; Nogueira et al., 2003), and by use of these methods, genes that are preferentially expressed in mature and immature sugarcane stem tissues have been identified (Casu et al., 2003, 2004). Further characterization of expression patterns of candidate genes identified by array analysis, for example, during development or in response to stress, is generally done by means of Northern blot analysis or real-time quantitative PCR after reverse transcription (RT-qPCR) (Klok et al., 2002). Northern blotting requires a large amount of RNA template and is laborious and timeconsuming (Zou et al., 2002). Therefore, the use of more sensitive and efficient methods, such as RT-qPCR, is desirable (Gachon et al., 2004). To date, there have been no reports of the use of RT-qPCR to analyze differential expression of genes in sugarcane. Product formation is monitored during each cycle of the RT-qPCR reaction (Saunders, 2004) to enable rapid and specific detection of the amplification product (Gachon et al., 2004). Expression of the target gene is usually normalized relative to that of a reference gene, which is either known or assumed to express stable levels of transcripts in most tissue or at least in the tissues being compared. A number of reference gene transcripts, such as those encoding the small or large subunits of ribosomal RNA (rRNA), 13-actin, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), have been used as reference genes in RT-qPCR analyses (Bustin, 2000). Transcripts of rRNA genes are present at levels several orders of magnitude greater than transcripts of protein-coding genes, and their measurement requires dilution of the sample if they are to be used as a reference for measurement of mRNA. Diluting samples can be inconvenient and introduces a potential source of error. A major assumption of real-time PCR analysis is that the reference gene is expressed at a constant level in all tissues and at all stages of development and should not be affected by the experimental treatment (Bustin, 2000). Although rRNA is considered a stable reference because of its abundance, this may not be the case with transcripts of other reference genes that are commonly used. The selection of an appropriate reference gene is very important for RTqPCR analysis in order to obtain consistent and reliable results. The use of reference genes that are expressed at equivalent levels relative to rRNA in diverse samples also makes it possible to assess differences in the absolute amount of transcripts of target genes in such samples. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes (including rRNA) at various stages of development and in various genotypes in the S a c c h a r u m complex. This has enabled
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us to develop and assess optimal RT-qPCR protocols to quantify the tissuespecific expression of sugarcane genes of interest. Materials and Methods
Plant materials Samples of cultivar Ql17 were taken from 7- to 8-month-old sugarcane plants. Plant materials were grown in a glasshouse in pots. The plants were harvested in 3 replicates, each containing 3 stalks growing in the same pot. Several samples were taken from each stalk: immature leaf roll; lamina from the last fully expanded leaf; stem from meristem down to internode 2; stem internodes 4-5, 7-8, 10-11, and 13-14; and roots, lnternode numbers are as described in Moore (1987). All parts, including fibrous tissue, were chopped into small pieces, put in 50-mL polypropylene tubes, and immediately snap-frozen in liquid N 2. All samples were then kept at -80~ Internodes 8-9 were also taken from the maturing stem of another cultivar (Q124), Saccharum spontaneum (SES106), Saccharum officinarum (IJ76-237), and Saccharum robustum (NG57-56). Plants were grown in pots or in soil outdoors in Townsville, Queensland (19~ 146~
RNA preparation Total RNA from sugarcane tissues was extracted by means of a CsC1 pad method as described by Sambrook et al. (1989). About 4 g (fresh mass) of each sample tissue was ground in liquid N 2. Ground tissues were added to a denaturing solution containing 4 M guanidium thiocyanate, 25 mM sodium citrate, 0.5% Nlaurylsarcosine, and 1% 13-mercaptoethanol. The mixture was blended with a homogenizer (Polytron, Kinematica) and then centrifuged at 3000g for 15 min at 4~ The supernatant was transferred to an ultracentrifuge tube containing 3 mL of 5.7 M CsC1. Samples were ultracentrifuged in a Beckman L8-70M ultracentrifuge with a SW40Ti rotor for 20 h at 23,500 rpm (~70,000g) at 20~ After the supernatant was removed, the resulting RNA pellet was dissolved in diethyl pyrocarbonate (DEPC)-treated water and transferred to a microcentrifuge tube. Total RNA was stored in aliquots at -80~ after the addition of 0.1 vol of 3 M sodium acetate (pH 5.2) and 3 vol of 100% ethanol. The concentration of RNA was quantified spectrophotometrically. Total RNA samples were then treated with DNase I following the protocol for RNA cleanup of the RNAeasy kit (Qiagen).
Synthesis of cDNA for real-time PCR The protocol followed for cDNA synthesis was that of Schenk et al. (2003) with slight modifications, with use of reagents supplied in the Superscript III FirstStrand Synthesis Kit (Invitrogen). To prepare the cDNA, approximately 0.459.5 p.L of total RNA (2 ~tg) was mixed with both 1 ~tL of random hexamer (50 ng/~tL) and 1 ~L of oligo(dT)20 (50 ~tM) primers and incubated at 70~ for 5 min. The reaction mix was immediately chilled on ice for 2 min, and cDNA synthesis was then undertaken by addition of the RT master mix, containing 4 ~tL of 5• RT buffer, 1 ~tL of 0.1 M 1,4-dithiothreitol, and 0.5 ~tL (200 U/~tL) of Superscript III reverse transcriptase. The reaction was adjusted to a total volume of
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20 ~tL with water and was incubated at 50~ for 50 min, and then for 15 rain at 70~ and was then cooled to 4~ All cDNA samples were tested by using standard PCR with the amounts corresponding to 100 ng of RNA, before further analysis. The amplification program was 95~ for 5 rain, followed by 35 cycles at 95~ for 30 s, 58~ for 30 s, and 72~ for 1 min, followed by a final step at 72~ for 7 min and then cooled to 4~ PCR products were separated by electrophoresis in a 3% agarose gel and visualized by staining with ethidium bromide. Predicted product sizes were verified with a 1-kb ladder of DNA markers before analysis by means of qPCR. For use as templates for qPCR, cDNA samples were diluted 10-fold in sterile water (Water for Injection BP, Pharmacia & UpJohn Company) for the assay of I]-actin, I]tubulin, and GAPDH transcripts (final concentration corresponding to 10 ng of RNA/~tL) and 10,000-fold for rRNA primers (10 pg RNA/~L).
Primer design Specific primers for reference and target genes were designed to conserved regions on the basis of comparison of sequences from several plants and sugarcane ESTs. Sequences from Arabidopsis, rice, maize, and sugarcane ESTs were obtained from the National Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov/) and also from the Institute for Genomic Research (http:// www.tigr.org/). The homology of gene sequences to sugarcane ESTs of each target gene was examined by a BLASTn search of the ESTs limited to Saccharum species (Altschul et al., 1990). The conserved regions of a particular gene from Arabidopsis and/or rice, maize, and sugarcane ESTs were obtained by aligning them with the Clustal X software (EMBL-EBI, European Bioinformatics Institute). Two primer pairs were designed and assessed for 2 different domains of the sugarcane 25S rRNA gene found in the database (25S rRNA1F-25S rRNA1R, 25S rRNA2F-25S rRNA2R). A single primer pair was designed for I]-actin (ActinFActinR), ~-tubulin (TubulinF-TubulinR), and GAPDH (GAPDHF-GAPDHR). The GAPDH primers were designed to the cytosolic form of the enzyme. The ~-actin forward primer was designed by use of information from the genomic sequence of rice to place it across a predicted intron (ActinF). All primers were designed with Primer Express software version 1.5 (Applied Biosystems) set to an annealing temperature of 58-60~ with an amplicon size of 100-120 bp. All primers were synthesized by GeneWorks Pty. Ltd., Australia.
RT-qPCR protocol The RT-qPCR analyses of reference and target gene transcripts in cDNA samples were conducted in an ABI model 7000 thermocycler (Applied Biosystems). A 25-~tL PCR reaction was prepared containing 5 ~tL of template cDNA, 5 ~tL of primer mix, 2.5 ~tL of water, and 12.5 ~tL of 2• SYBR green master mix (Applied Biosystems). The final primer concentration used for rRNA, GAPDH, and ~-actin primers was 0.2 p.M, and 0.6 ~tM was used for the [~-tubulin primers. The final amount of cDNA template assayed was equivalent to 50 ng of RNA for the protein-coding genes and 50 pg of RNA for rRNA. All samples were amplified in duplicate assays under the following conditions: 95~ for 10 min for 1 cycle,
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followed by 45 cycles of 95~ for 15 s and 1 min at 59~ The PCR products for each primer set were also subjected to melt-curve analysis. The melt-curve analysis was done from 60-95~ to ensure that the resulting fluorescence originated from a single PCR product and did not represent primer dimers formed during the PCR or a nonspecific product. No-template controls were also included to detect any spurious signals arising from amplification of any DNA contamination or primer dimer formed during the reaction. RT-qPCR results were analyzed with the sequence detection software SDS version 1.1 (Applied Biosystems). The SYBR green fluorescent signal was standardized to a passive reference dye (ROX) included in the PCR master mix (Edwards, 2004). Direct detection of the PCR product was measured by monitoring the increase in fluorescence caused by the binding of SYBR green dye to double-stranded DNA. A fluorescence threshold was set manually to a ARn of 0.3 on the log fluorescence scale to determine the fractional cycle number (Ct value) at which the fluorescence passed the detection threshold. For each cDNA sample, relative expression levels of each proteincoding gene were normalized by reference to the rRNA gene assay. The transcript abundance ratio of target gene to reference gene was determined by the following equation: relative expression = (Eref)Ctref/(Etarget) Cttarget (Pfaffl, 2001; Czechowski et al., 2004), where Eref and Etarget are the efficiencies of the primers for the reference and target gene, respectively, and Ctref and Cttarget are the mean Ct value of reference and target genes, respectively. The RT-qPCR protocol was optimized by determining the optimal primer concentrations and primer efficiencies. Several primer concentrations ranging from 0.1-0.6 ~tM were used to determine the optimal primer concentrations. Primer amplification efficiency was determined with data from amplification plots of individual reaction tubes generated during RT-qPCR. Fluorescence data generated from each reaction were exported into the LinRegPCR software version 7.5 (
[email protected]; Ramakers et al., 2003), which determined the slope of the exponential portion of the amplification curve of cycle number versus log fluorescence.
Application of the RT-qPCR protocol to measure the expression of a gene of interest A sugarcane transporter gene, Pst2a (GenBank accession no. AY165599), was used to assess the RT-qPCR protocols developed in this study. Two primers were designed to amplify a specific region of this gene: 5'-GTGCCCTGTTGGTTGGTATTG-3' (forward) and 5'-TGCCACACCAGCTTGCTC-3' (reverse). Real time qPCR assays (as described above) were used to measure abundance of Pst2a gene transcripts, with both rRNA and GAPDH used as reference genes. Results and Discussion
Development of the reverse transcription, followed by real-time quantitative PCR protocol (RT-qPCR) Determining reference gene primers and their optimal concentrations. All of the primers for reference genes were designed for conserved regions on the basis of
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Table 1. The sequence of primers used for reverse transcription followed by real-time quantitative PCR (RT-qPCR) assays of sugarcane cDNA, and the database designations of representative accessions of the sugarcane ESTs used for their design. Also listed are database designations of their identically matched homologs from Arabidopsis, rice, and maize. Primers Match
Primer Name 25S rRNA 25S rRNA 1F 25S rRNA1R 25S rRNA2F 25S rRNA2R -actin Actin F Actin R -tubulin TubulinF TubulinR GAPDH GAPDHF GAPDHR
Primer Sequences
ATAACCGCATCAGGTC TCCAAG CCTCAGAGCCAATCCT TTTCC GCAGCCAAGCGTTCAT AGC CCTATTGGTGGGTGAA CAATCC
Sugarcane EST
Arabidopsis
Rice
Maize
CO373883"
AY090986
M11585
AJ309824
CAI71131
AY090986
M11585
AJ309824
BQ536525
AY090986
Ml1585
AJ309824
BQ536525
AY090986
M11585
AJ309824
CTGGAATGGTCAAGG CTGGT TCCTTCTGTCCCATCC CTACC
CA148161
CCAAGTTCTGGGAGG TGATCTG TTGTAGTAGACGTTGA TGCGCTC
CA222437
CACGGCCACTGGAAG CA TCCTCAGGGTTCCTGA TGCC
AK063598
CA148161
AY103587
XM469133
L10634
CA222437
L10634
CA254672
X 07156
CA254672
X 07156
*The accession number is given for only one representative matching sequence on the NCBI database (http://www.ncbi.nlm.nih.gov/).
comparison of sequences from several plants and sugarcane ESTs (Table 1). Conserved regions suitable for designing PCR primers were identified from the alignment, and the primers were designed to fit the sugarcane sequence. Analysis of the rice genome database showed that [3-actin and ~-tubulin existed in gene families in monocots, and the sequences of the most abundant members of each family in the sugarcane EST database were selected to represent the sugarcane sequence. It should be noted that all primer pairs used showed a high degree of conservation between the sugarcane sequences and their rice and/or maize homologues and should have direct utility in other monocots. Because rRNA is the major component of purified RNA preparation and is unlikely to vary greatly as a proportion of total RNA (Sambrook et al., 1989), we compared 2 primer pairs targeted to 25S rRNA to determine which might serve as the best primary standard. Those primer pairs were designed for 2 different domains of the sugarcane 25S rRNA gene found in the database. The priming site for 25S rRNA1 is near the 5' end of the 25S rRNA gene and is close to the site for rRNA assays that have been used for various dicots, whereas the priming site for 25S rRNA2 is closer to the 3" end
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of the 25S rRNA gene. The ~-actin forward primer was designed by use of information from the genomic sequence of rice to place it across a predicted intron (ActinF). Experiments using 4 different primer concentrations (0.1, 0.2, 0.4, and 0.6 ~tM) demonstrated that for 25S rRNA, GAPDH, and [3-actin, a primer concentration of 0.2 ktM was found to be optimal because any further increase in primer concentration had little effect on the Ct value. However, for 13-tubulin, a concentration of 0.6 ~tM gave a lower Ct value than a concentration of 0.2 ~tM (data not shown), and the higher concentration was used for further work. These primer sets have also been tested for interference from any genomic DNA by comparing Ct values given by equivalent amounts of genomic DNA, cDNA after reverse transcription of RNA, and RNA that has not been reversetranscribed. The results showed very low levels of genomic DNA measured in the samples that had not been reverse-transcribed, indicating that these assays are not prone to interference when standard procedures are used for purifying and reverse transcribing RNA (data not presented). This was the case not only for primers designed over a splice junction as for ~-actin but also for the other primer pairs. Determining primer efficiency of assays for rRNA and protein-coding genes. The amplification efficiency of PCR primers for each of the reference gene assays (Table 1) was determined from the slope of the exponential phase of individual amplification plots of independent sets of amplification data within an experiment designed to compare tissue-specific differences in gene expression (ARn versus cycle number) by use of the LinRegPCR software version 7.5. The 2 assays both had a mean E value of 1.9 (_+0.06 SD) for 25S rRNA1 and 25S rRNA2. Because Northern blotting is the traditional method of following gene expression and this depends largely on rRNA to determine equal loading of RNA in wells, we use rRNA as the primary standard for our RT-qPCR. On the basis of accumulated PCR efficiency data, we conclude that either of the 2 rRNA assays could be used as a primary reference for estimating the relative expression of protein-coding genes. Determining primer efficiency and sensitivity of assays for protein-coding genes showed that high values for PCR efficiency were obtained for the GAPDH and I]-actin assays, with E values of 1.93 (_+0.09 SD) and 1.94 (_+0.11 SD), respectively. However, ~-tubulin assays gave a lower E value of 1.81 (_+0.08 SD).
Comparing reference gene expression in different sugarcane tissues To assess expression of the reference genes in different tissues, we obtained total RNA samples from different parts of the sugarcane plant from 3 biological replications as described in "Methods." RNA from each sample was reversetranscribed and assayed in duplicate by means of RT-qPCR (Figure 1). Expression of rRNA. Figure 1 showed that rRNA has the most consistent expression in different parts of sugarcane tissues, compared with that of GAPDH, [~-actin, and ~-tubulin. The 2 rRNA assays we used seem to have differences in their Ct values. Ct values give a direct indication of the sensitivity of detection of a cDNA template. Because we used highly purified RNA obtained by CsC1 density centrifugation, we expected to get almost identical Ct values for the rRNA assays of different biological samples. However, results varied, and the 25S rRNA1 assay gave Ct values ranging from 18.7-21.9 (mean _+ SD, 19.8 -+ 0.8) for the 8
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25S rRNA
GAPDH
g
6
~
4 2 0
-~
5
"~
4
n~
~-Actin
3 2 1 0
13-Tubulin
3 2 1 0 LR
LFE
M
14-5 17-8 110-11113-14 R
Tissue Figure 1. Relative expression of reference genes in various sugarcane tissues. Assays of transcript levels for each reference gene were normalized by use of the 25S rRNAI assay to determine ACt values and, hence, ratios of gene expression relative to the large ribosomal RNA (rRNA) subunit. Relative expression values for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 13-actin, and 13tubulin should be multiplied by 1000 to give actual relative expression levels. Error bars indicate standard errors of the mean for each tissue (n = 3). LR, leaf roll; LFE, last fully expanded leaf; M, meristem; 14-5, internodes 4-5; ~7-8, internodes 7-8; I10-I!, internodes I0-t 1; II3-14, interr~odes 1314; R, root.
tissue types, each sampled in triplicate. Similarly, the 25S rRNA2 assay gave Ct values ranging from 16.6-21.0 (mean _+ SD, 18.2 _+ 1.2). Thus, the Ct values varied over 3-4 PCR cycles over the 3 biological replicates, which might represent a combination of the intrinsic technical variability of the RT-qPCR protocol and biologically variability. We attribute this technical variability to uncontrolled factors such as errors in estimating total RNA (spectrophotometry errors and impurities of absorbance at 260 nm) and variable efficiency in different reverse transcription reactions. These data showed that, averaged across all tissue samples, the 25S rRNA2 assay was more sensitive and gave a lower mean Ct vaiue than did the
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25S rRNA1 assay. Because both assays were targeted to the same 25S rRNA, this indicated that the 25S rRNA2 primers were approximately 3 times more sensitive than the 25S rRNA1 primers. Figure 1 shows that this difference in sensitivity was remarkably similar for the different tissue types. Possible causes of the difference in sensitivity include differences in RNA secondary structure at the 2 priming sites, resulting in different yields of cDNA from the same amount of initial rRNA; different SYBR green binding abilities of the amplicons produced by each assay; and differences in rRNA sequence at priming sites for these 2 assays in the rRNA genes originating from the different species (S. officinarum and S. spontaneum), contributing to the interspecific hybrid Q117 genome (given the highly conserved nature of 25S rRNA genes or the primer sequences selected [Table 1], we believe this latter possibility is unlikely). This phenomenon was not studied further. rRNA is abundant, requiring a 1:1000 dilution of total RNA samples to enable a direct comparison with amplification plots of protein-coding genes. Therefore, it was more convenient to choose the less-sensitive 25S rRNA1 assay as a reference for determining relative expression of the protein-coding genes. For further experiments, 25S rRNA1 was chosen as the primary rRNA reference assay. _Expression of protein-coding genes. PCR primers for the protein-coding genes listed in Table 1 were tested to determine the abundance of their corresponding mRNAs relative to rRNA (25S rRNA 1 assay) in different parts of the sugarcane plant. Results presented in Figure 1 indicate that GAPDH displayed considerably less variation across different tissues than did 13-actin and 13-tubulin. This was also shown by their calculated coefficients of variation, which were 51%, 68%, and 81% for GAPDH, [3-actin, and ~-tubulin, respectively. Furthermore, assuming equal efficiencies of reverse transcription and equal fluorescence signal per PCR amplicon, GAPDH transcripts were on average the most abundant of the protein-coding genes tested. Ribosomal RNA, [3-actin, 13-tubulin, and GAPDH represent housekeeping genes that are commonly used as references to normalize patterns of gene expression via Northern analysis or RT-qPCR (Bustin, 2000). Ideally, a reference gene should be expressed at a relatively constant level in different tissues and at all stages of development (Bustin, 2000). On the basis of the data presented above, GAPDH transcripts appear to show more constant expression across different sugarcane tissues than do ~-actin and ~-tubulin genes. GAPDH is an abundant glycolytic enzyme present in most cell types (Giulietti et al., 2001) and has been widely used as a reference gene in RT-PCR experiments in animal and human samples. However, several reports in human and animal systems have suggested that this housekeeping gene had limitations for use as an internal control because of variation in tissue-specific expression and variability resulting from various experimental treatments (Bustin, 2000; Giulietti et al., 2001). Because rRNA represents most of the total RNA present in a cell, it is considered to be a reliable internal control or reference gene, proven in many experiments quantifying the level of transcripts from animal, human, and plant tissues (Bustin, 2000; Edwards, 2004). The use of rRNA as a reference gene for real-time PCR in rice demonstrated its consistency of expression compared with that of GAPDH, [~-actin, and ~-tubulin under different stages of seed development,
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different cultivars, and different levels of UV irradiation treatment (Kim et al., 2003). However, the high abundance of rRNA requires greater template dilutions to bring its measurement in cDNA samples within the dynamic range of RTqPCR. These dilution steps require more work and impose a further level of potential error, adding to inaccuracy in the PCR analysis. Consequently, it would be advantageous to have another protein-coding gene that is expressed at a level similar to genes of interest for use as a reference gene. From the results of relative expression across different parts of the sugarcane plant, the most appropriate gene of those tested herein would be GAPDH. It should be noted, however, that GAPDH varied in abundance approximately 4-fold in different tissues relative to 25S rRNA: for example, it was most abundant in the last fully expanded leaf and internode 10-11 samples and least abundant in the internode 13-14 and root samples. Expression of reference genes across cultivars and species Commercial sugarcane cultivars are mostly derived from the hybridization of S. officinarum L. with a wild relative S. spontaneum L. (Grivet and Arruda, 2001). Some other species, such as S. robustum, Saccharum sinense, and Saccharum barberi, have also contributed germplasm in the breeding of sugarcane cultivars (Aitken et al., in press). Other Saccharum species have potential as a source of novel genes for future introgression into sugarcane. Therefore, expression of the reference housekeeping genes noted above was investigated in another sugarcane cultivar and other selected Saccharam species by means of our RT-qPCR protocol. RNA samples from 2 commercial cultivars and 3 Saccharum species were analyzed by RT-qPCR as described previously. Consistent with the previous assays of Ql17 (Figure 1), each of the other genotypes gave higher values of expression relative to the 25S rRNA1 assay for GAPDH, followed by 13-actin, with much lower levels of [3-tubulin (data not shown). Figure 2 shows values for relative expression averaged across all of these assays and shows that [3-actin and GAPDH had lower coefficients of variation (31% and 33%, respectively) than did ~-tubulin (47%) when data from all the above genotypes were analyzed. Taken together with the higher relative expression values, this suggests that 13-actin and GAPDH represent better reference genes for comparing expression across sugarcane cultivars and/or Saccharum species and in most situations would be comparable to the rRNA assays as reference genes. Application of the RT-qPCR protocol to evaluate the expression of a sugar transporter gene The expression of the sugar transporter gene Pst2a in sugarcane tissues relative to rRNA varies widely (Figure 3). The gene was expressed at a very low level in leaf and meristem tissues. In contrast, the transporter gene was relatively highly expressed in the stem internodes 4-11. However, the transporter gene expression was reduced at lower internodes (internodes 13-14) and expressed even less in the root. In general, the relative expression of Pst2a to the GAPDH reference gene showed a pattern similar to that of rRNA (Figure 3) but differed somewhat in
335
Real-time qPCR in sugarcane
3. 'o 33% 2.
"~ rr-
1
GAPDH
Actirl
Tubulin
Reference genes
Figure 2. Expression of reference genes in mature stem tissue relative to ribosomal RNA (rRNA) (25S rRNA1 assay) across genotypes of sugarcane and related species. Error bars indicate standard error of the mean for all genotypes (n = 5). The numbers above bar charts indicate coefficients of variation. e?, o
18 16
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Tissue Figure 3. Relative expression of the transporter gene Pst2a with respect to the expression of 2 other reference genes in 8 sugarcane tissues. (A) Pst2a expression with respect to ribosomal RNA (rRNA) (25S rRNA1) gene; (B) Pst2a expression with respect to the expression of glyceraldehyde-3phosphate dehydrogenase (GAPDH). Tissues used in this experiment were the same as those used in Figure 1. Error bars indicate standard error of the means (n = 3).
internodes 13-14 and root, where GAPDH is expressed at lower levels than is rRNA (Figure 1). This result compares well with Pst2a transcript analysis by Northern blot reported by Casu et al. (2003), which demonstrated that the expression of Pst2a was highly abundant in maturing stem tissue but no expression was
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detected in mature leaf or roots. However, the RT-qPCR method described herein was more sensitive in detecting gene expression than the Northern blot analysis, it allowed quantification of relative expression, and was more amenable to biological replication and statistical analysis. These experiments demonstrate the utility of the proposed reference genes (GAPDH and rRNA) in studies of developmentally regulated gene expression in sugarcane. However, addressing other biological issues, such as biotic and abiotic stress responses, will require these genes to be tested to determine which is best suited to the experimental conditions.
Conclusion We have developed assays for RT-qPCR analysis of sugarcane to compare GAPDH, [~-tubulin, and 13-actin as reference genes, in addition to the commonly used 25S rRNA gene. Expression levels of 25S rRNA and GAPDH were higher and more consistent across tissues compared with [3-actin and ~-tubulin. 25S rRNA was the most sensitive and consistent as a reference gene, but cDNA samples require more dilution than do samples of protein-coding genes. For the range of tissue types examined during this investigation, therefore, GAPDH appears to be the best protein-coding reference gene of those tested for normalization of gene expression measured by RT-qPCR in sugarcane. The RT-qPCR protocols described herein will have important applications in quantifying expression levels of genes of potential functional interest selected from functional genomics studies, as exemplified by results obtained on the putative sugar transporter gene Pst2a.
Acknowledgments We thank Dr Anne Rae, Mayling Goode, Mark Jackson, and Donna Glassop for providing some RNA samples and technical help in this research. H.M. Iskandar acknowledges the support of an AusAID Scholarship.
References Aitken KS, Jackson PA, and McIntyre CL (in press) A combination of AFLP and SSR markers provides extensive map coverage and identification of homo(eo)logous linkage groups in a sugarcane cultivar. Theor Appl Genet. Altschul SE Gish W, Miller W, Meyers EW, and Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403-410. Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25: 169-193. Carson DL and Botha FC (2000) Preliminary analysis of expressed sequence tags for sugarcane. Crop Sci 40: 1769-1779. Casu RE, Grof CPL, Rae AL, McIntyre CL, Dimmock CM, and Manners JM (2003) Identification of a novel sugar transporter homologue strongly expressed in maturing stem vascular tissues of sugarcane by expressed sequence tag and microarray analysis. Plant Mol Biol 52: 371-386. Casu RE, Dimmock CM, Chapman SC, Grof CPL, Mclntyre CL, Bonnett GD, and Manners JM (2004) Identification of differentially expressed transcripts from maturing
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