Abstract
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used to analyze the relative gene expression level, however, the accuracy of qRT-PCR is greatly affected by the stability of reference genes, which is tissue- and environment- dependent. Therefore, choosing the most stable reference gene in a specific tissue and environment is critical to interpret gene expression patterns. Aluminum (Al), cadmium (Cd), and heat stresses are three important abiotic factors limiting soybean (Glycine max) production in southern China. To identify the suitable reference genes for normalizing the expression levels of target genes by qRT-PCR in soybean response to Al, Cd and heat stresses, we studied the expression stability of ten commonly used housekeeping genes in soybean roots and leaves under these three abiotic stresses, using five approaches, BestKeeper, Delta Ct, geNorm, NormFinder and RefFinder. We found TUA4 is the most stable reference gene in soybean root tips under Al stress. Under Cd stress, Fbox and UKN2 are the most stable reference genes in roots and leaves, respectively, while 60S is the most suitable reference gene when analyzing both roots and leaves together. For heat stress, TUA4 and UKN2 are the most stable housekeeping genes in roots and leaves, respectively, and UKN2 is the best reference gene for analysis of roots and leaves together. To validate the reference genes, we quantified the relative expression levels of six target genes that were involved in soybean response to Al, Cd or heat stresses, respectively. The expression patterns of these target genes differed between using the most and least stable reference genes, suggesting the selection of a suitable reference gene is critical for gene expression studies.
Highlights
Quantitative reverse transcription polymerase chain reaction is one of the most widely used techniques to detect the changes in gene expression [1], due to its relatively accurate quantification, high sensitivity and high throughput
Housekeeping genes are commonly used as the reference genes for Quantitative reverse transcription polymerase chain reaction (qRT-PCR), such as 18S ribosomal RNA (18S rRNA), 25S ribosomal RNA (25S rRNA), β-actin (ACT11), cyclophilin (CYP2), elongation factor 1-alpha (ELF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor (TEF), tubulin (TUB4) and polyubiquitin (UBQ) [4]
In order to verify the performance of the candidate reference genes identified in this study, six target genes were selected for qRT-PCR to study their gene expression under Al, Cd and heat stresses, in which GmALMT1 [35] and GmARI1 [36] have been reported to play roles in soybean tolerance to Al toxicity, GmHMA13 and GmHMA19 were related to Cd stress [37], and GmGBP1 and GmHsfA1 were responsive to heat stress [27, 38]
Summary
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is one of the most widely used techniques to detect the changes in gene expression [1], due to its relatively accurate quantification, high sensitivity and high throughput. The difference in the amount and quality of the template can affect the efficiency of the qRT-PCR reactions [2], it is essential to normalize the expression level of the target gene by using a reference gene as an internal control. An ideal reference gene should demonstrate a consistent expression level across all tested tissues or conditions [3]. Many recent studies found that the expression levels of housekeeping genes may vary largely across different tissues, developmental stages, or experimental conditions [6,7,8,9]. It is necessary to select stably expressed reference genes before they are utilized for normalizing the expression levels of target genes by qRT-PCR
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
