Dataset Information

Quantitative real-time PCR analysis of four genetically modified soybean events using plasmid and genomic DNA calibrators.

ABSTRACT: A calibration curve is required for reliable and accurate quantitative real-time PCR analysis of genetically modified (GM) organisms, necessitating the use of reference materials as calibrators. In this study, two types of DNA calibrators-plasmid DNA (pDNA) and genomic DNA (gDNA)-were used to quantify four GM soybean events (SYHT0H2, MON87751, DAS-44406-6, and DAS-81419-2). The PCR efficiency and linearity for the calibrators adhered to the CODEX guidelines. The conversion factor (Cf) was calculated as the ratio of copies of GM events to those of endogenous genes using the pDNA calibration curve. To assess the accuracy and repeatability of these assays, quantification at GM levels of 3% and 1% was performed. Based on our results, we believe that the pDNA calibrator assessed in this study can be used as a reference material for GMO quantitative analysis and can replace gDNA, especially considering the ease of management and advantages of mass production.

Supplementary information

The online version contains supplementary material available at 10.1007/s10068-023-01392-0.

SUBMITTER: Shin MK

PROVIDER: S-EPMC10866821 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Quantitative real-time PCR analysis of four genetically modified soybean events using plasmid and genomic DNA calibrators.

Shin Min Ki MK Jeon Seon Min SM Park Jong Seok JS

Food science and biotechnology 20230811 4

A calibration curve is required for reliable and accurate quantitative real-time PCR analysis of genetically modified (GM) organisms, necessitating the use of reference materials as calibrators. In this study, two types of DNA calibrators-plasmid DNA (pDNA) and genomic DNA (gDNA)-were used to quantify four GM soybean events (SYHT0H2, MON87751, DAS-44406-6, and DAS-81419-2). The PCR efficiency and linearity for the calibrators adhered to the CODEX guidelines. The conversion factor (Cf) was calcul ...[more]

PMID: 38371688

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Project description:BackgroundNormalizing to housekeeping gene (HKG) can make results from quantitative real-time PCR (qRT-PCR) more reliable. Recent studies have shown that no single HKG is universal for all experiments. Thus, a suitable HKG should be selected before its use. Only a few studies on HKGs have been done in plants, and none in soybean, an economically important crop. Therefore, the present study was conducted to identify suitable HKG(s) for normalization of gene expression in soybean.ResultsAll ten HKGs displayed a wide range of Ct values in 21 sample pools, confirming that they were variably expressed. GeNorm was used to determine the expression stability of the HGKs in seven series sets. For all the sample pools analyzed, the stability rank was ELF1B, CYP2 > ACT11 > TUA > ELF1A > UBC2 > ACT2/7 > TUB > G6PD > UBQ10. For different tissues under the same developmental stage, the rank was ELF1B, CYP2 > ACT2/7 > UBC2 > TUA > ELF1A > ACT11 > TUB > G6PD > UBQ10. For the developmental stage series, the stability rank was ACT2/7, TUA > ELF1A > UBC2 > ELF1B > TUB > CYP2 > ACT11 > G6PD > UBQ10. For photoperiodic treatments, the rank was ACT11, ELF1B > CYP2 > TUA > ELF1A > UBC2 > ACT2/7 > TUB > G6PD > UBQ10. For different times of the day, the rank was ELF1A, TUA > ELF1B > G6PD > CYP2 > ACT11 > ACT2/7 > TUB > UBC2 > UBQ10. For different cultivars and leaves on different nodes of the main stem, the ten HKGs' stability did not differ significantly. Delta Ct approach and 'Stability index' were also used to analyze the expression stability in all 21 sample pools. Results from Delta Ct approach and geNorm indicated that ELF1B and CYP2 were the most stable HKGs, and UBQ10 and G6PD the most variable ones. Results from 'Stability index' analysis were different, with ACT11 and CYP2 being the most stable HKGs, and ELF1A and TUA the most variable ones.ConclusionOur data suggests that HKGs are expressed variably in soybean. Based on the results from geNorm and Delta Ct analysis, ELF1B and CYP2 could be used as internal controls to normalize gene expression in soybean, while UBQ10 and G6PD should be avoided. To achieve accurate results, some conditions may require more than one HKG to be used for normalization.

Project description:According to the Canadian Food Inspection Agency and Health Canada, genetically modified crops are considered safe if they are substantially equivalent to a conventional crop in regards to agronomic, physiological and compositional characteristics. A recurring issue in safety assessment of genetically modified crops is the paucity of analytical methods to detect unintended or unexpected outcomes of genetic modification. Traditional targeted compound comparative analyses are limited in scope and capacity to detect unintended changes in chemical composition. This study explored the potential of using microarray technology to assess the substantial equivalence of gene expression profiles between genetically modified and conventional soybean cultivars. Different pre processing methods were applied to the raw expression data from the arrays, and clustering methods were used to try and differentiate the genetically modified cultivars from the conventional cultivars. Results showed that more variation existed between different strains of conventional cultivars than between conventional and genetically modified cultivars. For more information, please see: Cheng, K.C., Beaulieu, J., Iquira, E., Belzile, F.J., Fortin, M.G. and StrÃ¶mvik, M.V. (2008). âEffect of transgenes on global gene expression in soybean is within the natural range of variation of their conventional counterparts.â Journal of Agricultural and Food Chemistry (in press) Experiment Overall Design: Five samples (biological replicates) of total RNA from each of the five different soybean varieties were selected for hybridization to Affymetrix Soybean GeneChips, for a total of 25 chips (following total RNA integrity assessment). Spike controls B2, bio-B, bio-C, bio-D and Cre-x were added to each hybridization cocktail. Arrays were washed and stained in an Affymetrix Fluidics Station prior to scanning on the Affymetrix GeneChip Scanner 3000. Image acquisition and processing was done with the Affymetrix Microarray Analysis Suite 5.0.

Dataset Information

Quantitative real-time PCR analysis of four genetically modified soybean events using plasmid and genomic DNA calibrators.

Supplementary information

Publications

Quantitative real-time PCR analysis of four genetically modified soybean events using plasmid and genomic DNA calibrators.

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