title: Validation of housekeeping genes as an internal control for gene expression studies in Giardia lamblia using quantitative real-time PCR.
abstract: The analysis of transcript levels of specific genes is important for understanding transcriptional regulation and for the characterization of gene function. Real-time quantitative reverse transcriptase PCR (RT-qPCR) has become a powerful tool to quantify gene expression. The objective of this study was to identify reliable housekeeping genes in Giardia lamblia. Twelve genes were selected for this purpose, and their expression was analyzed in the wild type WB strain and in two strains with resistance to nitazoxanide (NTZ) and metronidazole (MTZ), respectively. RefFinder software analysis showed that the expression of the genes is different in the three strains. The integrated data from the four analyses showed that the NADH oxidase (NADH) and aldolase (ALD) genes were the most steadily expressed genes, whereas the glyceraldehyde-3-phosphate dehydrogenase gene was the most unstable. Additionally, the relative expression of seven genes were quantified in the NTZ- and MTZ-resistant strains by RT-qPCR, using the aldolase gene as the internal control, and the results showed a consistent differential pattern of expression in both strains. The housekeeping genes found in this work will facilitate the analysis of mRNA expression levels of other genes of interest in G. lamblia.
authors.affiliation: CONACyT Research Fellow-Instituto Nacional de Pediatría, Secretaría de Salud, 04530, México. Electronic address: jmarcialqu@conacyt.mx.
authors.forename: Jaime
authors.initials: J
authors.lastname: Marcial-Quino
authors.affiliation: Laboratorio de Ingeniería Genética y Metabolitos Secundarios, Departamento de Biotecnología, Universidad Autónoma Metropolitana-Iztapalapa, 09340, México. Electronic address: degfff@yahoo.com.
authors.forename: Francisco
authors.initials: F
authors.lastname: Fierro
authors.affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud, 04530, México. Electronic address: ignaciodelamora@yahoo.com.mx.
authors.forename: Ignacio
authors.initials: I
authors.lastname: De la Mora-De la Mora
authors.affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud, 04530, México. Electronic address: sergioenr@gmail.com.
authors.forename: Sergio
authors.initials: S
authors.lastname: Enríquez-Flores
authors.affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud, 04530, México. Electronic address: saulmanzo@ciencias.unam.mx.
authors.forename: Saúl
authors.initials: S
authors.lastname: Gómez-Manzo
authors.affiliation: Laboratorio de Neurociencias, Instituto Nacional de Pediatría, Secretaría de Salud, 04530, México. Electronic address: america_vc@yahoo.com.mx.
authors.forename: America
authors.initials: A
authors.lastname: Vanoye-Carlo
authors.affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud, 04530, México. Electronic address: itzheltorres@hotmail.com.
authors.forename: Itzhel
authors.initials: I
authors.lastname: Garcia-Torres
authors.affiliation: Colegío de Ciencias y Humanidades, Plantel Casa Libertad, Universidad Autónoma de la Ciudad de Mexico, 09620, México. Electronic address: edgar.sierra@uacm.edu.mx.
authors.forename: Edgar
authors.initials: E
authors.lastname: Sierra-Palacios
authors.affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud, 04530, México. Electronic address: hreyesvivas@yahoo.com.mx.
authors.forename: Horacio
authors.initials: H
authors.lastname: Reyes-Vivas
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D017931:DNA Primers; D005786:Gene Expression Regulation; D020043:Genes, Essential; D016829:Giardia lamblia / Q000187:drug effects / Q000235:genetics*; D008795:Metronidazole / Q000494:pharmacology; D009574:Nitro Compounds; D060888:Real-Time Polymerase Chain Reaction; D013844:Thiazoles / Q000494:pharmacology
chemical_list: D000981:Antiprotozoal Agents; D017931:DNA Primers; D009574:Nitro Compounds; D013844:Thiazoles; D008795:Metronidazole; C041747:nitazoxanide
keywords: G. lamblia; Gene expression; Housekeeping genes; RT-qPCR; RefFinder program; Validation