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.

Distribution and function of 3′,5′-Cyclic-AMP phosphodiesterases in the human ovary

“…Simultaneous isolation of mRNA and native protein, first strand cDNA synthesis and realtime quantitative PCR was performed as described previously (Petersen and Andersen, 2014). TaqMan® Gene Expression Assays (pre-designed) for qPCR were purchased from Life Technologies (Table S2).…”

“…The activity of the different PDE enzyme families was measured as previously described (Petersen and Andersen, 2014). The contribution of the different family members were evaluated by measuring the difference in activity with the addition of following specific inhibitors to the reaction buffer: trequinsin 20 nM (PDE3, IC50: …”

Phosphodiesterases in the rat ovary: effect of cAMP in primordial follicles

“…Ovaries were isolated from day 4 post-natal (PN) Han-wistar rats (Harlan, Horst, The Netherlands) as previously published (Petersen & Andersen 2014). For isolation of GC, 10-week-old Wistar rats (Taconic, Ejby, Denmark) were stimulated with 22.5 IU FSH/LH (Menopur, Ferring, Copenhagen, Denmark) and two groups were injected after 46 h with 20 IU human chorionic gonadotropin (hCG) (Pregnyl, MSD, Ballerup, Denmark).…”

“…RT was carried out using the High capacity cDNA kit (Life Technologies). Real-time quantitative PCR was performed as described previously (Petersen & Andersen 2014) with TaqMan primer probe assays (Assay ID) against Pde1b (Rn00575591_m1), Pde1c (Rn00579334_m1),…”

“…The roflumilast sensitive PDE4 activity was determined by subtracting the roflumilast-insensitive activity from the total activity. The PDE assays were performed using a cAMP-PDE SPA assay as detailed previously (Petersen & Andersen 2014) with the following modifications: 0.1% BSA (Sigma-Aldrich) was used in the reaction buffer and the activity was measured once after 15 min.…”