In-depth analysis of the endogenous reference genes used in the quantitative PCR detection systems for rice

Abstract

To standardize the rice-specific PCR detection methods, five previously reported rice (Oryza sativa) taxon-specific genes were compared and evaluated. The investigated genes included the rice root-specific gene (gos9), the ppi phosphofructokinase gene (ppi-PPF), the phospholipase D gene (PLD), the starch branching enzyme gene (RBE4) and the sucrose phosphate synthase gene (SPS). Sequencing analyses revealed that among the tested rice cultivars, single-nucleotide polymorphisms (SNPs) existed in the gos9, PLD, ppi-PPF and SPS amplicons, though no statistically significant effect on their Ct values was found. The ppi-PPF and PLD systems were found to produce amplicons in non-rice species, such as sugarcane and broomcorn. The quantitative real-time PCR results revealed that this cross-reaction led to an underestimate of the GM (genetically modified) rice content. With the exception of the aforementioned shortcomings, these five endogenous reference genes all have acceptable amplification efficiencies, which ranged from 98 to 108 %, and high sensitivity within the limit of detection (LOD) values, which ranged from 5 to 10 copies of the haploid genome. In estimating the GM content in blinded rice samples, these five systems produce relatively accurate quantitative results with deviations less than 15 %, but the RBE4 system produced the most accurate quantitative results. Therefore, we have determined that the RBE4 gene is the most suitable rice reference gene, and the gos9 and SPS genes can also be used as rice reference genes because they have good commutability with the RBE4 gene. Care should be taken when interpreting results based on the PLD and ppi-PPF genes owing to their cross-reaction with other species.

Development, Optimization, and Single Laboratory Validation of an Event-Specific Real-Time PCR Method for the Detection and Quantification of Golden Rice 2 Using a Novel Taxon-Specific Assay

“…22 However, two studies evidenced the presence of technical problems for most of those assays, confirmed by additional bioinformatics analyses performed by the MBG Unit of the IHCP, which in many cases led to method underperformance with respect to amplification efficiency or assay specificity. 23,24 In view of this, together with IRRI, the MBG Unit of the IHCP thus decided to develop and validate in-house a new taxonspecific assay for rice, designed on the PLD α2 gene and named PLD-GR, and an event-specific assay for GR2. Primers and probes for the two assays were designed by IRRI, while bioinformatics analyses, assay optimization, characterization, and in-house validation were conducted by the MBG Unit of the IHCP with the samples provided by IRRI.…”

“…The taxon-specific assay, targeting an endogenous gene, is very important for correct quantification. − To act as a reliable reference for GMO quantification, a taxon-specific assay must be specific; its sequence must be present in single copy in the plant’s genome and stably amplified across different varieties of the target species. , Different rice taxon-specific assays were available for GMO analysis at the time when this project was started: ppi Phosphofructokinase (ppi-PPF), RBE4 and gos9, − sucrose phosphate synthase (SPS), , and Phospholipase D (PLD) developed by Bayer . However, two studies evidenced the presence of technical problems for most of those assays, confirmed by additional bioinformatics analyses performed by the MBG Unit of the IHCP, which in many cases led to method underperformance with respect to amplification efficiency or assay specificity. , In view of this, together with IRRI, the MBG Unit of the IHCP thus decided to develop and validate in-house a new taxon-specific assay for rice, designed on the PLD α2 gene and named PLD-GR, and an event-specific assay for GR2. Primers and probes for the two assays were designed by IRRI, while bioinformatics analyses, assay optimization, characterization, and in-house validation were conducted by the MBG Unit of the IHCP with the samples provided by IRRI.…”

Selection of reference genes for flowering pathway analysis in the masting plants, Celmisia lyallii and Chionochloa pallens, under variable environmental conditions

“…A list of reference gene sequences was prepared based on previous reports of reference gene selection for plants in either the Asteraceae or Poaceae. Gene sequences encoding elongation factor 1-α ( EF - 1α ) 60 , glyceraldehyde - 6 - phosphate dehydrogenase ( GAPDH ) 20 , eukaryotic initiation factor 4ε ( eIF - 4ε ) 34 , phosphoglycerate kinase 1 ( PGK1 ) 61 , protein phosphatase 2a ( PP2a ) 61 , metalloprotease ( MTP ) 61 , SAND 61 , actin ( ACT ) 62 , β - tubulin ( TUB ) 30 , expressed protein ( ExP ) 63 , nuclease - binding protein ( NBP ) 63 , and tumour homolog protein ( THP ) 63 were downloaded from the Genbank database and used to search for the corresponding homologous sequences in the Celmisia and Chionochloa transcriptomes (Table 1). Candidate reference gene sequences were identified from the transcriptomic data (unpublished) using TBLASTN 64 .…”