Defining Suitable Reference Genes for RT-qPCR Analysis on Intestinal Epithelial Cells

The study of the mammalian intestinal epithelium concerns several aspects of cellular and molecular biology. In fact, most of these studies aim to define molecular components or mechanisms related with the control of stemness and the balance between cell proliferation and differentiation in physiopa...

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Vydáno v:	Molecular biotechnology Ročník 54; číslo 3; s. 930 - 938
Hlavní autoři:	Sirakov, Maria, Borra, Marco, Cambuli, Francesca Maria, Plateroti, Michelina
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	New York Springer-Verlag 01.07.2013 Humana Press Inc Springer Nature B.V
Témata:	Animals Biochemistry Biological Techniques Biotechnology Cell Biology Cell Line cell proliferation Cellular biology Chemistry Chemistry and Materials Science computer software cytology epithelial cells Epithelial Cells - cytology Epithelial Cells - physiology Gene Expression Genes Genetic Markers Genetic Markers - genetics genetics Human Genetics intestinal mucosa Intestinal Mucosa - cytology Intestinal Mucosa - physiology messenger RNA methods Mice Molecular biology pathophysiology physiology Polymerase chain reaction Protein Science Rats Reference Values reverse transcriptase polymerase chain reaction Reverse Transcriptase Polymerase Chain Reaction - methods Reverse Transcriptase Polymerase Chain Reaction - standards Ribonucleic acid RNA Rodents Small intestine Intestinal crypt Reference gene Intestinal epithelium IEC6 RT-qPCR
ISSN:	1073-6085, 1559-0305, 1559-0305
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Abstract	The study of the mammalian intestinal epithelium concerns several aspects of cellular and molecular biology. In fact, most of these studies aim to define molecular components or mechanisms related with the control of stemness and the balance between cell proliferation and differentiation in physiopathological conditions. It is worth mentioning that real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) approaches are commonly used, but only a few studies are available regarding suitable reference genes to normalize gene expression data. The present study was designed to validate potential reference genes in freshly isolated proliferating or differentiated epithelial cells from the mouse intestine. We also extended our analysis to the IEC6 intestinal epithelial cells, as a promising model to study intestinal physiopathology in vitro. The stability of six potential reference genes (Hprt1, Ppia, Gapdh, Rplp0, Ppib, and Vil1) has been tested both in epithelial cells isolated from the mouse intestine and in the IEC6 cell line. The software programs—geNorm and Normfinder—were used to obtain an estimation of the expression stability of each gene and, by comparing the results, to identify the most suitable genes for RT-qPCR data normalization. These multiple approaches allowed us to select different suitable reference genes for the correct quantification of mRNAs depending on the differentiated or proliferative nature of the cells.
AbstractList	The study of the mammalian intestinal epithelium concerns several aspects of cellular and molecular biology. In fact, most of these studies aim to define molecular components or mechanisms related with the control of stemness and the balance between cell proliferation and differentiation in physiopathological conditions. It is worth mentioning that real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) approaches are commonly used, but only a few studies are available regarding suitable reference genes to normalize gene expression data. The present study was designed to validate potential reference genes in freshly isolated proliferating or differentiated epithelial cells from the mouse intestine. We also extended our analysis to the IEC6 intestinal epithelial cells, as a promising model to study intestinal physiopathology in vitro. The stability of six potential reference genes (Hprt1, Ppia, Gapdh, Rplp0, Ppib, and Vil1) has been tested both in epithelial cells isolated from the mouse intestine and in the IEC6 cell line. The software programs—geNorm and Normfinder—were used to obtain an estimation of the expression stability of each gene and, by comparing the results, to identify the most suitable genes for RT-qPCR data normalization. These multiple approaches allowed us to select different suitable reference genes for the correct quantification of mRNAs depending on the differentiated or proliferative nature of the cells. The study of the mammalian intestinal epithelium concerns several aspects of cellular and molecular biology. In fact, most of these studies aim to define molecular components or mechanisms related with the control of stemness and the balance between cell proliferation and differentiation in physiopathological conditions. It is worth mentioning that real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) approaches are commonly used, but only a few studies are available regarding suitable reference genes to normalize gene expression data. The present study was designed to validate potential reference genes in freshly isolated proliferating or differentiated epithelial cells from the mouse intestine. We also extended our analysis to the IEC6 intestinal epithelial cells, as a promising model to study intestinal physiopathology in vitro. The stability of six potential reference genes (Hprt1, Ppia, Gapdh, Rplp0, Ppib, and Vil1) has been tested both in epithelial cells isolated from the mouse intestine and in the IEC6 cell line. The software programsâ[euro]"geNorm and Normfinderâ[euro]"were used to obtain an estimation of the expression stability of each gene and, by comparing the results, to identify the most suitable genes for RT-qPCR data normalization. These multiple approaches allowed us to select different suitable reference genes for the correct quantification of mRNAs depending on the differentiated or proliferative nature of the cells.[PUBLICATION ABSTRACT] The study of the mammalian intestinal epithelium concerns several aspects of cellular and molecular biology. In fact, most of these studies aim to define molecular components or mechanisms related with the control of stemness and the balance between cell proliferation and differentiation in physiopathological conditions. It is worth mentioning that real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) approaches are commonly used, but only a few studies are available regarding suitable reference genes to normalize gene expression data. The present study was designed to validate potential reference genes in freshly isolated proliferating or differentiated epithelial cells from the mouse intestine. We also extended our analysis to the IEC6 intestinal epithelial cells, as a promising model to study intestinal physiopathology in vitro. The stability of six potential reference genes (Hprt1, Ppia, Gapdh, Rplp0, Ppib, and Vil1) has been tested both in epithelial cells isolated from the mouse intestine and in the IEC6 cell line. The software programs-geNorm and Normfinder-were used to obtain an estimation of the expression stability of each gene and, by comparing the results, to identify the most suitable genes for RT-qPCR data normalization. These multiple approaches allowed us to select different suitable reference genes for the correct quantification of mRNAs depending on the differentiated or proliferative nature of the cells.The study of the mammalian intestinal epithelium concerns several aspects of cellular and molecular biology. In fact, most of these studies aim to define molecular components or mechanisms related with the control of stemness and the balance between cell proliferation and differentiation in physiopathological conditions. It is worth mentioning that real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) approaches are commonly used, but only a few studies are available regarding suitable reference genes to normalize gene expression data. The present study was designed to validate potential reference genes in freshly isolated proliferating or differentiated epithelial cells from the mouse intestine. We also extended our analysis to the IEC6 intestinal epithelial cells, as a promising model to study intestinal physiopathology in vitro. The stability of six potential reference genes (Hprt1, Ppia, Gapdh, Rplp0, Ppib, and Vil1) has been tested both in epithelial cells isolated from the mouse intestine and in the IEC6 cell line. The software programs-geNorm and Normfinder-were used to obtain an estimation of the expression stability of each gene and, by comparing the results, to identify the most suitable genes for RT-qPCR data normalization. These multiple approaches allowed us to select different suitable reference genes for the correct quantification of mRNAs depending on the differentiated or proliferative nature of the cells. The study of the mammalian intestinal epithelium concerns several aspects of cellular and molecular biology. In fact, most of these studies aim to define molecular components or mechanisms related with the control of stemness and the balance between cell proliferation and differentiation in physiopathological conditions. It is worth mentioning that real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) approaches are commonly used, but only a few studies are available regarding suitable reference genes to normalize gene expression data. The present study was designed to validate potential reference genes in freshly isolated proliferating or differentiated epithelial cells from the mouse intestine. We also extended our analysis to the IEC6 intestinal epithelial cells, as a promising model to study intestinal physiopathology in vitro. The stability of six potential reference genes ( Hprt1, Ppia, Gapdh, Rplp0, Ppib, and Vil1 ) has been tested both in epithelial cells isolated from the mouse intestine and in the IEC6 cell line. The software programs—geNorm and Normfinder—were used to obtain an estimation of the expression stability of each gene and, by comparing the results, to identify the most suitable genes for RT-qPCR data normalization. These multiple approaches allowed us to select different suitable reference genes for the correct quantification of mRNAs depending on the differentiated or proliferative nature of the cells.
Author	Cambuli, Francesca Maria Plateroti, Michelina Borra, Marco Sirakov, Maria
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CitedBy_id	crossref_primary_10_1371_journal_pone_0086033 crossref_primary_10_1038_s41419_022_04607_0 crossref_primary_10_1007_s12257_025_00211_z crossref_primary_10_1016_j_molmet_2017_07_009 crossref_primary_10_3390_cells10061555 crossref_primary_10_1038_s41598_018_29139_6 crossref_primary_10_1038_s41467_023_40993_5 crossref_primary_10_1111_jpn_13126
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Snippet	The study of the mammalian intestinal epithelium concerns several aspects of cellular and molecular biology. In fact, most of these studies aim to define...
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SubjectTerms	Animals Biochemistry Biological Techniques Biotechnology Cell Biology Cell Line cell proliferation Cellular biology Chemistry Chemistry and Materials Science computer software cytology epithelial cells Epithelial Cells - cytology Epithelial Cells - physiology Gene Expression Genes Genetic Markers Genetic Markers - genetics genetics Human Genetics intestinal mucosa Intestinal Mucosa - cytology Intestinal Mucosa - physiology messenger RNA methods Mice Molecular biology pathophysiology physiology Polymerase chain reaction Protein Science Rats Reference Values reverse transcriptase polymerase chain reaction Reverse Transcriptase Polymerase Chain Reaction - methods Reverse Transcriptase Polymerase Chain Reaction - standards Ribonucleic acid RNA Rodents Small intestine
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Title	Defining Suitable Reference Genes for RT-qPCR Analysis on Intestinal Epithelial Cells
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