Simultaneous Stabilization of Protein and RNA from a Single Tissue Homogenate

Retaining the activity and stability of proteins and RNA extracted from biological samples remains a major concern and challenge during sample preparation and storage. Many complicated and time-consuming strategies have been devised to preserve important and costly biological samples, but a universal method has yet to be identified.

Common Challenges with Protein and RNA Extraction from Tissue Samples

  • Samples not compatible with secondary analysis
  • Loss of sample integrity with long-term storage and freeze-thaw cycles

Sample preparation methods are typically dictated by downstream analysis but selecting the extraction protocol necessary for the target analytical method often hampers the ability to use samples again in secondary analysis. Biological samples are routinely frozen for long-term storage, but depending on the preparation method, sample integrity can change over time making sample archiving infeasible. Additionally, certain analytes lose functional activity after freezing and thawing, preventing repeat analyses including determining correlations between protein and nucleic acid precursors.

To address these issues, we developed Stabilyser™ reagent – a single-step collection, lysis and multi-analyte stabilization reagent that preserves protein activity and RNA stability (Figure 1). When prepared using Stabilyser™ reagent, samples contain a uniform mixture of proteins and RNA that can be used directly in a variety of downstream applications. Additionally, samples can withstand multiple freeze-thaw cycles, eliminating issues with sample integrity during long-term storage and repeat analysis. In this article, we will demonstrate the utility of adding Stabilyser™ reagent to your sample preparation and purification methods, improving both RNA and protein enzyme stability.

Workflow chart describing the procedure of protein and RNA isolation using Stabilyser™ reagent. The procedure requires no modifications to your sample preparation methods, other than the addition of Stabilyser™ reagent during tissue homogenization/cell lysis. The addition of this reagent allows for the use of the sample in multiple downstream analysis methods.

Figure 1. Procedure for the isolation of proteins and RNA from tissue samples, using Stabilyser™ reagent. The addition of this reagent requires no changes in sample preparation protocol, while providing the ability to perform multiple types of downstream analysis.

Experimental Methods for the Evaluation of Stabilyser™ Reagent

Three different experiments were performed to demonstrate the utility of Stabilyser™ reagent in RNA and protein enzyme stability. For controls, tissue samples were also lysed using traditional reagents, such as PBS or CytoBuster™ reagent. Specific methods for each experiment can be found in the corresponding section below.

RNA Stability

Whole mouse kidneys, livers and brains were lysed in 5 mL of PBS (1X, pH 7.4), CytoBuster™ reagent, or Stabilyser™ reagent. All samples were clarified by centrifugation. Protein quantification was performed using a Pierce A660nm assay and samples were adjusted to a concentration of 1 mg/mL prior to being aliquoted and stored at -80 ⁰C. Lysates repeatedly underwent freeze-thaw cycles using liquid nitrogen and a 37 ⁰C water bath, respectively, prior to RNA purification. RNA was purified with a GenElute™ Total RNA Miniprep kit, using the following modified protocol:

100 µL of the lysate was diluted into 350 µL of the provided Lysis Solution/2-ME buffer, followed by the addition of 250 µL of 200 proof ethanol.  Samples were loaded onto the spin column and purified following the rest of the manufacturer’s protocol.

RNA stability was monitored using an Agilent 2100 BioAnalyzer with the Agilent RNA 6000 Nano Kit.

Lysis Efficiency and Protein Enzyme Stability

250 mg sections of chicken hearts were lysed in 5 mL of CytoBuster™ reagent or Stabilyser™ reagent. Protein quantification was performed using a Pierce A660nm assay and samples were adjusted to a concentration of 1 mg/mL prior to storage at -20 ⁰C. Overall protein yield was assessed by SDS-PAGE. Samples were normalized and loaded at 10 μg protein per lane. Enzymatic activity was monitored using an IDH activity assay kit after each freeze-thaw cycle. Samples were frozen at -20 ⁰C for two days prior to thawing the sample to measure enzymatic activity.

Comparison of Stabilyser™ Reagent with Traditional Cell Lysis Reagents

RNA Stability

After lysis with PBS, CytoBuster™ reagent, or Stabilyser™ reagent, RNA was purified using the GenElute™ Total RNA kit with a modified protocol (see methods). After purification, RNA stability for each lysate was assessed using an Agilent 2100 BioAnalyzer (Figure 2). In comparison to other cell lysis reagents, Stabilyser™ reagent provided RNA stability throughout the lysis and purification process, as seen by the lack of degradation products found in each final sample.

This is an image of an Agilent BioAnalyzer Gel of purified RNA samples from each mouse tissue type (kidney, liver, and brain) and each cell lysis reagent (PBS, Cytobuster™ reagent, and Stabilyser™ reagent). PBS shows no RNA degradation for brain tissue, minimal degradation for kidney tissue, and moderate degradation for liver tissue. Cytobuster™ reagent shows no degradation for brain tissue and severe degradation for both kidney and liver tissue. In contrast, Stabilyser™ reagent shows no appreciable degradation in any of the tissue types.

Figure 2. Comparison of RNA activity of each lysate after purification. In comparison to other reagents, samples collected using Stabilyser™ reagent show no degraded RNA products.

Following initial analysis, RNA samples are commonly frozen for storage, then thawed for secondary analysis. To demonstrate improvements in stability with Stabilyser™ reagent, purified RNA samples from each tissue type were subjected to four freeze-thaw cycles (Figure 3). Regardless of the tissue type, samples prepared using Stabilyser™ reagent showed no degradation after the first two cycles and minimal degradation in after the third and fourth cycles.

This is an image of an Agilent BioAnalyzer Gel of purified RNA samples of mouse kidney, liver, and brain that were isolated using Stabilyser™ reagent. All three samples were subjected to four freeze-thaw cycles, and RNA stability was measured after each thaw cycle. Regardless of the tissue type, samples prepared using Stabilyser™ reagent showed no degradation after the first two cycles and minimal degradation in after the third and fourth cycles.

Figure 3. Comparison of RNA activity after multiple freeze-thaw cycles.

Lysis Efficiency

As Stabilyser™ reagent is compatible with a variety of mechanical homogenizers, using Stabilyser™ reagent requires no modifications to tissue homogenization protocols. To ensure Stabilyser™ reagent provides the same lysis efficiency as other reagents without modification, overall protein yield was compared using three different cell lysis reagents. Chicken heart sections were homogenized using either PBS, CytoBuster™ reagent, or Stabilyser™ reagent, and overall protein yield was measured using SDS-PAGE. Stabilyser™ reagent provided higher protein extraction yields, with five-times more protein than PBS buffer, and two-times more protein than CytoBuster™ reagent. Additionally, comparing samples from each lysate with normalized protein concentration shows that high molecular weight proteins appear to be extracted at a greater efficiency (Figure 4). While this has yet to be tested in all possible tissue types, this may provide a potential additional benefit for researchers using Stabilyser™ reagent.

This image of an SDS-PAGE gel compares the molecular weights of proteins from three different cell lysates. Cell lysates were prepared using chicken hearts and either PBS, Cytobuster™ reagent, or Stabilyser™ reagent. Samples were normalized and the gel was loaded with 10 μg of protein per lane. The molecular weight distribution of proteins from lysates isolated using PBS or Cytobuster™ reagent were relatively the same, but the lysate from Stabilyser™ reagent showed an increased concentration of high molecular weight proteins.

Figure 4. Molecular weight comparison of proteins from each lysate using SDS-PAGE. Normalized and loaded at 10 μg of protein per lane

 

Protein Enzyme Assay

Typically frozen for storage after initial analysis, protein samples commonly display reduced protein activity in secondary analysis. To demonstrate improved protein stability with Stabilyser™ reagent, the enzymatic activity of proteins found in lysates prepared with either CytoBuster™ reagent or Stabilyser™ reagent was measured after several freeze-thaw cycles (Figure 5).

This image compares the enzymatic activity of proteins isolated from chicken hearts after three freeze-thaw cycles. Cell lysates were isolated using either Cytobuster™ or Stabilyser™ reagent and enzymatic activity was measured by IDH activity.  Proteins isolated using Stabilyser™ reagent retained similar activity over all freeze-thaw cycles, while proteins from Cytobuster™ reagent showed a 20 unit decrease in activity after a single freeze-thaw cycle.

Figure 5. Evaluation of protein enzyme activity by IDH activity after each freeze-thaw cycle

Conclusions

Stabilyser™ reagent yields enhanced stability and activity of both protein and RNA extracted from homogenized tissue, as demonstrated by the comparison with other cell lysis reagents. Tissue samples homogenized using Stabilyser™ reagent and stored under frozen conditions maintained similar RNA levels and protein enzyme activity through multiple freeze-thaw cycles. Stabilyser™ reagent enables the user to obtain both genetic and protein information from a single sample, significantly alleviating laborious sample preparation.

General Considerations when Using Stabilyser™ Reagent

  • Variable results may occur when trying to compare protein and RNA processed from different tissue sections. This problem can be addressed by using a single homogenous lysate to compare different analytes.
  • Greater instability can occur in some tissue types, as demonstrated above in mouse tissues. While general extraction buffers may work for a specific tissue or application, they are not considered universal. The Stabilyser™ reagent provides reliable results across many tissue types.
  • Different extraction buffers may yield different amounts of protein and nucleic acids and should be selected carefully for your tissue type and method.

The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.

Stabilyser™, CytoBuster™, and GenElute™ are trademarks of Merck KGaA, Darmstadt, Germany and/or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.

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