title: Simultaneous isolation of mRNA and native protein from minute samples of cells.
abstract: Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native proteins from samples containing small numbers of cells. Our approach utilizes oligodeoxythymidylate [oligo(dT)25]-coated paramagnetic beads in an optimized reaction buffer to isolate mRNA comparable in quantity and quality to mRNA isolated with existing methods, while maintaining the proteins in their native state for traditional protein assays. We validated the procedure using neonatal rat ovaries and small numbers of human granulosa cells, demonstrating the extraction of mRNA suitable for gene expression analysis with simultaneous isolation of native proteins suitable for downstream characterization using different protein assays.
authors.affiliation: Laboratory of Reproductive Biology, The Juliane Marie Centre for Women, Children, and Reproduction - Copenhagen University Hospital, Copenhagen University, Denmark; LEO Pharma, Ballerup, Denmark.
authors.forename: Tonny Studsgaard
authors.initials: TS
authors.lastname: Petersen
authors.affiliation: Laboratory of Reproductive Biology, The Juliane Marie Centre for Women, Children, and Reproduction - Copenhagen University Hospital, Copenhagen University, Denmark.
authors.forename: Claus Yding
authors.initials: CY
authors.lastname: Andersen
mesh_terms: D000818:Animals; D053148:Caspase 3 / Q000235:genetics; D005260:Female; D006107:Granulosa Cells; D006801:Humans; D007265:Inhibins / Q000235:genetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics; D008967:Molecular Biology / Q000295:instrumentation / Q000379:methods*; D010053:Ovary / Q000166:cytology; D011489:Protein Denaturation; D011506:Proteins / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000302:isolation & purification*; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012269:Ribosomal Proteins / Q000235:genetics; D059467:Transcriptome
chemical_list: D011506:Proteins; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C094377:inhibin-alpha subunit; C041806:ribosomal protein L32; D007265:Inhibins; D007770:L-Lactate Dehydrogenase; D053148:Caspase 3
keywords: RNA isolation and purification; enzyme assay; oligo (dT); ovary