title: Purification of Single-Stranded cDNA Based on RNA Degradation Treatment and Adsorption Chromatography.
abstract: Analysis of gene expression is a common research tool to study networks controlling gene expression, the role of genes with unknown function, and environmentally induced responses of organisms. Most of the analytical tools used to analyze gene expression rely on accurate cDNA synthesis and quantification to obtain reproducible and quantifiable results. Thus far, most commercial kits for isolation and purification of cDNA target double-stranded molecules, which do not accurately represent the abundance of transcripts. In the present report, we provide a simple and fast method to purify single-stranded cDNA, exhibiting high purity and yield. This method is based on the treatment with RNase H and RNase A after cDNA synthesis, followed by separation in silica spin-columns and ethanol precipitation. In addition, our method avoids the use of DNase I to eliminate genomic DNA from RNA preparations, which improves cDNA yield. As a case report, our method proved to be useful in the purification of single-stranded cDNA from the pathogenic fungus Sporothrix schenckii.
authors.affiliation: a Departamento de Biología , División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta s/n, col. Noria Alta , Guanajuato , Gto. , México.
authors.forename: Elías
authors.initials: E
authors.lastname: Trujillo-Esquivel
authors.affiliation: a Departamento de Biología , División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta s/n, col. Noria Alta , Guanajuato , Gto. , México.
authors.forename: Bernardo
authors.initials: B
authors.lastname: Franco
authors.affiliation: a Departamento de Biología , División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta s/n, col. Noria Alta , Guanajuato , Gto. , México.
authors.forename: Alberto
authors.initials: A
authors.lastname: Flores-Martínez
authors.affiliation: a Departamento de Biología , División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta s/n, col. Noria Alta , Guanajuato , Gto. , México.
authors.forename: Patricia
authors.initials: P
authors.lastname: Ponce-Noyola
authors.affiliation: a Departamento de Biología , División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta s/n, col. Noria Alta , Guanajuato , Gto. , México.
authors.forename: Héctor M
authors.initials: HM
authors.lastname: Mora-Montes
mesh_terms: D002853:Chromatography, Liquid; D018076:DNA, Complementary / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000302:isolation & purification*; D020871:RNA Stability; D012331:RNA, Fungal / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D013175:Sporothrix
chemical_list: D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D012331:RNA, Fungal; D012259:Ribonuclease, Pancreatic
keywords: Single-stranded cDNA purification; Sporothrix schenckii; gene expression analysis