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In-depth analysis of the endogenous reference genes used in the quantitative PCR detection systems for rice
Ist Teil von
European food research & technology, 2012-06, Vol.234 (6), p.981-993
Ort / Verlag
Berlin/Heidelberg: Springer-Verlag
Erscheinungsjahr
2012
Link zum Volltext
Quelle
SpringerLink (Online service)
Beschreibungen/Notizen
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.