Simultaneous RNA and protein extraction from the same experimental sample represents a fast-growing demand for researchers who are interested in gene regulation mechanisms. Studies of gene regulation at the mRNA and protein levels are often complicated by small sample sizes and the need to use disparate techniques to obtain nucleic acid and protein. Dividing a sample into several fractions for independent purification procedures is difficult, especially for small samples (e.g., biopsy material). Even more problematic is the difficulty in interpreting experimental results if the samples used for nucleic acid and protein purifications are not identical.

The NucleoSpin RNA/Protein kit allows the concurrent isolation of total RNA and protein from a single sample with no division required, making it especially valuable for unique, small, and/or precious samples. Isolated RNA is suitable for all common downstream applications, with RNA quality and purity comparable to stand-alone RNA purification kits. Isolated protein may be directly used in SDS-PAGE and Western blot analyses.

Figure 1. Extraction of RNA and protein from a single sample using a simple workflow.

During the NucleoSpin RNA/Protein procedure, cells are lysed by incubating in lysis buffer containing a large amount of chaotropic ions. The lysis buffer immediately inactivates virtually all enzymes (e.g., RNases, proteases, and phosphatases) that are present in almost all biological materials. The lysis buffer brings even low-solubility proteins into solution and creates appropriate binding conditions to promote the adsorption of RNA to the silica membrane. Under these conditions, protein from all cellular compartments easily passes through the specially treated NucleoSpin RNA/Protein column. The denaturing properties of the lysis buffer eliminate the need for expensive and harmful proteinase inhibitors.

Protein is precipitated from this first flowthrough with the Protein Precipitator (PP) buffer. After a wash step, the protein pellet is dissolved in Protein Solving Buffer (PSB) containing TCEP, an odorless reducing agent. The protein can then be readily used in SDS-PAGE analysis. Contaminating genomic DNA is digested on-column with an RNAse-free recombinant DNase (rDNase, supplied with the kit) which is applied directly to the silica membrane during the workflow. A series of simple washing steps removes salts, metabolites, and macromolecular cellular components. Finally, pure RNA is eluted with RNase-free water.

NucleoSpin RNA/Protein provides high-quality RNA

Figure 2. RNA prepared by the NucleoSpin RNA/Protein kit is of high quality. RNA was extracted from 10⁶ HeLa cells using the NucleoSpin RNA/Protein kit and eluted from the column using 100 μl of RNase-free water. 1 μl was analyzed on an Agilent Bioanalyzer. An RNA trace and digital electropherogram (inset) reveal high-quality RNA with minimal degradation.

Efficient protein isolation using NucleoSpin RNA/Protein

Figure 3. Efficient isolation of protein from a variety of sample types using the NucleoSpin RNA/Protein kit. Total protein was isolated from a variety sample types using the NucleoSpin RNA/Protein kit, subjected to SDS-PAGE, and stained with Coomassie Blue. Gels depicted contain a protein ladder (M), 10⁶ HeLa cells (A), 30-mg liver samples (B), and 100-mg garden cress seedling samples (C). 1.4% of total isolated protein was loaded per lane, indicating quantitative isolation across all sample types.

Expression analysis of cleaved caspase-3 and Erk2 in carcinoma cell lines upon treatment with a DNA-damaging agent that induces apoptosis

The following data was kindly provided by Steffen Naumann and Prof. B. Kaina, Department of Toxicology, University of Mainz, Germany.

To demonstrate that the DNA-damaging agent induced apoptosis, cells were stimulated for 24, 48, and 120 hours and assayed for cleaved caspase-3 expression.

Figure 4. Treatment of a carcinoma cell line with a DNA-damaging agent induces apoptosis. NucleoSpin RNA/Protein was used to isolate protein from carcinoma cell lines that were either untreated (Lane 1) or treated for 24 hr (Lane 2), 48 hr (Lane 3), or 120 hr (Lane 4) with a DNA-damaging agent to induce apoptosis. Protein fractions were analyzed via Western blots probed for cleaved caspase-3 (Panel A) and a loading control (Erk2) (Panel B), revealing an increase in cleaved caspase-3 protein levels in the treated samples.

Inducible knock-down of Hint1 in MCF-7 Tet-On cells

To demonstrate that the protein Hint1 is involved in modulation and expression of p53, Hint1 was silenced by previously validated short hairpin RNAs (shRNAs).

Figure 5. shRNA-mediated knockdown of Hint1 induces commensurate reductions in p53 protein and mRNA levels. In cells stably transfected with empty vector (mock), functional shRNA against Hint1 (shRNA-Hint1 430/431), and inactive shRNA against Hint1 (shRNA-Hint1 432/433), expression was induced with 1 μg/ml doxycycline. RNA and protein were isolated from the same undivided samples using the NucleoSpin RNA/Protein kit. After 48 hr, 50 μg of protein was analyzed by Western blotting with anti-Hint1 (A) or anti-p53 (B) antibody with α-actinin used as a loading control. RT-PCR analysis of RNA derived from the same samples as A and B demonstrated a significant reductions in p53 mRNA levels in cells expressing functional shRNA against Hint1 (C).