2LEO Pharma, Ballerup, Denmark
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.
In many areas of biological research (e.g., reproductive biology), the amount of sample material is often limited, especially for human cells and tissues such as ovarian follicles or embryonic stem cells. It is often desirable to perform multiple assays on such samples—gene expression and protein analyses, for example. However, separate RNA and protein isolation steps are currently necessary to perform both types of assays from a single sample, which may not be possible for samples containing very few cells.
Protocols have been developed for simultaneous purification of RNA, proteins, and DNA (1) from small samples. However, most of these methods lead to protein denaturation in order to protect the RNA from enzymatic degradation, thereby precluding the use of assays that require proteins in their native state, such as enzymatic activity assays, native PAGE or co-immunoprecipitation.
The present study describes the development of a new method based on an existing denaturing magnetic bead oligo-dT protocol (www.lifetechnologies.com/dk/en/home/references/protocols/nucleic-acid-purification-and-analysis/mrna-protocols/dynabeads-mrna-direct-kit.html) to simultaneously isolate mRNA and proteins in their native state while reducing RNA degradation by carrying out the RNA isolation at 4°C in the presence of a reducing agent. This strategy has previously been used to inhibit the degradation of RNA (2, 3), either by disulfide bridge reduction in RNases (4) or activation of endogenous RNase inhibitors (5). The new protocol was developed using neonatal rat ovaries, human granulosa cells (hGCs), and human corpus luteum (CL) by measuring the mRNA expression of the inhibin alpha subunit (Inha) gene along with two housekeeping genes, the 60S ribosomal protein L32 (Rpl32) and glyceraldehyde 3-phosphate dehydrogenase (Gapdh), while measuring the enzymatic activities of phosphodiesterases (PDE), caspase-3, lactate dehydrogenase (LDH) and the immunoprecipitation of GAPDH and β-actin.Materials and methods Animal tissue
Ovaries were isolated from Han-Wistar rats (Harlan Laboratories, Horst, The Netherlands) post-natal day 7 ± 1. The animals were decapitated and the ovaries and oviducts were removed and placed in 1 mL McCoy-5A media (Life Technologies, Nærum, Denmark) at 38°C. The ovaries were transferred to 3 mL MEM-Alpha (Life Technologies) to avoid a HEPES-buffered media and then cleaned, followed by a brief wash in PBS (Life Technologies). The average weight per ovary was 0.4 ± 0.1 mg (mean ± SEM). The ovaries were snap-frozen in liquid nitrogen and stored at -80°C until RNA isolation.Human tissue
Corpus luteum biopsies for immunoprecipitation were obtained from women undergoing fertility preservation by having ovarian tissue cryopreserved (6). Granulosa cells from small antral follicles were used for measurement of caspase-3activity (7). The use of human tissue was approved by the ethics committee (H-2–2011–044). Human foreskin fibroblasts (hFFs) (ATCC, Manassas, VA) were used to determine the optimal salt concentration for mRNA isolation.
Buffers and solutions used for RNA extraction Our method allows for isolation of mRNA while retaining functional protein from samples containing small numbers of cells using oligo-dT-coated paramagnetic beads. The yield and quality of the extracted mRNA were comparable to that from existing RNA isolation methods, and the enzymatic activity in the lysate was preserved.
Our method allows for isolation of mRNA while retaining functional protein from samples containing small numbers of cells using oligo-dT-coated paramagnetic beads. The yield and quality of the extracted mRNA were comparable to that from existing RNA isolation methods, and the enzymatic activity in the lysate was preserved.
All reagents used were purchased from Sigma-Aldrich (Copenhagen, Denmark) unless otherwise stated. Buffers include: PDE lysis buffer: 50 mM Tris-HCl pH 7.5, 1M NaCl (Merck, Glostrup, Denmark), 6 mM CaCl2 (Merck), 8.3 mM MgCl2, 1.7 mM EGTA, 0.05% Triton-X 100, 1% Protease Inhibitor Cocktail for use with mammalian cell and tissue extracts; LDH lysis buffer: 50 mM Tris-HCl, pH 8.5, 1M NaCl (Merck), 1% Protease Inhibitor Cocktail, 5 mM dithiothreitol (DTT) (Apollo Scientific Ltd, Cheshire, UK), 0.1% Triton-X 100; Caspase-3 lysis buffer: 10 mM Tris-HCl, pH 7.5, 0.5M NaCl, 1 mM EDTA, 5 mM DTT, 0.1% Triton-X 100; LiDS lysis buffer: 100 mM Tris-HCl pH 7.5, 500 mM LiCl, 10 mM EDTA, 1% lithium dodecyl sulfate (LiDS), 5 mM DTT; Washing buffer A: 10 mM Tris-HCl, pH 7.5, 150 mM LiCl, 1 mM EDTA, 0.1% LiDS; Washing buffer B: 10 mM Tris-HCl, pH 7.5, 150 mM LiCl, 1 mM EDTA; 10× RT Buffer: 500 mM Tris-HCl pH 8.3, 750 mM KCl (Merck), 30 mM MgCl2; Elution buffer: 10 mM Tris-HCl, pH 7.5.