ACTB and GAPDH appear at multiple SDS-PAGE positions, thus not suitable as reference genes for determining protein loading in techniques like Western blotting.
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ABSTRACT:
SUBMITTER: Zhang K
PROVIDER: S-EPMC8669867 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The genes encoding β-actin (ACTB in human or Actb in mouse) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH in human or Gapdh in mouse) are the two most commonly used references for sample normalization in determination of the mRNA level of interested genes by reverse transcription (RT) and ensuing polymerase chain reactions (PCR). In this study, bioinformatic analyses revealed that the ACTB, Actb, GAPDH and Gapdh had 64, 69, 67 and 197 pseudogenes (PGs), respectively, in the corresponding genome. Most of these PGs are intronless and similar in size to the authentic mRNA. Alignment of several PGs of these genes with the corresponding mRNA reveals that they are highly homologous. In contrast, the hypoxanthine phosphoribosyltransferase-1 gene (HPRT1 in human or Hprt in mouse) only had 3 or 1 PG, respectively, and the mRNA has unique regions for primer design. PCR with cDNA or genomic DNA (gDNA) as templates revealed that our HPRT1, Hprt and GAPDH primers were specific, whereas our ACTB and Actb primers were not specific enough both vertically (within the cDNA) and horizontally (compared cDNA with gDNA). No primers could be designed for the Gapdh that would not mis-prime PGs. Since most of the genome is transcribed, we suggest to peers to forgo ACTB (Actb) and GAPDH (Dapdh) as references in RT-PCR and, if there is no surrogate, to use our primers with extra caution. We also propose a standard operation procedure in which design of primers for RT-PCR starts from avoiding mis-priming PGs and all primers need be tested for specificity with both cDNA and gDNA.
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Project description:BackgroundCapparis spinosa L. is a typical desert plant that is resistant to high temperatures and drought, and at the same time is rich in medicinal and food values. The objective of this study is to explore the variations in nutrient composition, morphological characteristics, and SDS-PAGE patterns of caper seeds from different provenances, aiming to provide insights for the selection of superior seed provenances.ResultsIn this experiment, there were significant differences in the morphological characteristics and major nutritional components of caper seeds from different provenances. Seeds from the YKL (Karayagaqi Township, Yining County) and YKG (G218, KashiTown, Yining County) regions were larger in size compared to seeds from other regions. Among the four measured nutritional components, crude fat had the highest content, especially in the YKL and YKG region. The results of correlation analysis showed that crude fat was negatively correlated with soluble sugar and soluble protein but significantly positively correlated with starch content. As longitude increased from east to west, the morphological characteristics gradually increased. Based on the principal component analysis of all the parameters of the seeds, the eight provenances could be classified into three groups. HM (Hami), TGS (S202, Gaochang District, Turpan), HYW (Wubao Town, Yizhou District, Hami), TQQ (Qiquanhu Town, Turpan), and TLF (Turpan) were a group with higher soluble protein, soluble sugar, and water content. YKL and YKG were in one group, which had larger seed grains with high crude fat and starch content. AKS (Aksu) was in a separate group. The protein fractions from seeds of eight regions were extracted using Osborne fractionation method, it was found that glutelin content was the highest, while albumin content was the lowest. After these proteins were analyzed by SDS-PAGE, the electrophoretic patterns showed that the protein molecular weights were relatively small, and there were differences in protein bands among different provenances.ConclusionAccording to the PCA results, the eight seed provenances could be divided into three groups. There were both geographically distant ones clustered into one group, and those close to each other were also divided into one group. There were differences in seed morphology, nutrient content and SDS-PAGE profiles among the different seed sources. This difference might be caused by a combination of geographic and climatic factors. In addition, YKL and YKG were roughly selected as good seed provenances, which provided a theoretical basis for the development of C. spinosa L. germplasm resources.
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