Stem cell differentiation with stable genes represented as a tree of life.

Decoding Stem Cells: How to Pick the Right Genes for Accurate Research

Theo Raines in Science & Nature December 2025 • 4 min read.

"Unlock reliable stem cell research: A guide to stable reference genes in bone marrow-derived mesenchymal stromal cell differentiation."

Mesenchymal stromal cells (MSCs) are crucial for regenerative medicine due to their ability to transform into various cell types like bone, cartilage, and fat. This makes them a promising tool for treating injuries and diseases. However, the way MSCs behave and their molecular characteristics can vary depending on where they're sourced from (bone marrow, adipose tissue, etc.) and the specific culture conditions used in the lab.

Scientists often use quantitative polymerase chain reaction (qPCR) to measure gene expression in MSCs. qPCR is a sensitive technique, but its accuracy hinges on using reliable reference genes. Reference genes act as internal controls to normalize data and account for variations between samples. The problem? Commonly used reference genes can fluctuate under different experimental conditions, leading to misleading results.

This article breaks down recent research that tackles this challenge, identifying the most stable reference genes for bone marrow-derived MSCs (BM-MSCs) as they differentiate into fat and bone cells. By understanding these findings, researchers can improve the accuracy of their experiments and gain a clearer picture of MSC behavior.

The Key to Reliable Results: Finding Stable Reference Genes

Stem cell differentiation with stable genes represented as a tree of life.

The study meticulously evaluated nineteen potential reference genes in BM-MSCs undergoing differentiation into adipocytes (fat cells) and osteoblasts (bone cells). These genes were assessed using specialized software (NormFinder and GeNorm) to determine their expression stability under various conditions. The goal was to pinpoint genes that maintain consistent expression levels, regardless of the differentiation stage.

The research involved several key steps:

Isolating and Culturing BM-MSCs: MSCs were extracted from bone marrow samples and cultured in the lab. These cells were then induced to differentiate into either adipocytes or osteoblasts using specific culture mediums.
Characterizing MSCs: The researchers confirmed that the isolated cells possessed typical MSC characteristics by analyzing their surface markers and their ability to differentiate into the desired cell lineages.
Measuring Gene Expression: qPCR was used to measure the expression levels of the nineteen reference genes in undifferentiated MSCs, adipocytes, and osteoblasts.
Analyzing Stability: The data obtained from qPCR was analyzed using NormFinder and GeNorm software to rank the reference genes based on their expression stability.

The study revealed that no single reference gene was perfectly stable across all conditions. However, specific genes demonstrated superior stability depending on the experimental setup. For instance, when analyzing all three groups (undifferentiated, adipocytes, and osteoblasts) together, PBGD was identified as the most stable, closely followed by G6PDH. However, due to low expression levels of PBGD and G6PDH, RPLPO emerged as a more suitable option because of its higher expression with reasonably good stability.

Takeaways: Choosing the Right Gene for Your Experiment

This research emphasizes the importance of carefully selecting reference genes for qPCR experiments involving BM-MSCs. Using unstable reference genes can lead to inaccurate results and misinterpretations of MSC behavior.

Here’s a quick guide to help you choose:

<ul><li>For general studies involving undifferentiated, adipogenic, and osteogenic cells: Consider RPLPO due to its high expression and reasonable stability.</li><li>For studies comparing adipogenic and undifferentiated cells: PBGD and G6PDH show good stability.</li><li>Always validate your reference gene choices: Use software like NormFinder and GeNorm to assess stability in your specific experimental conditions.</li></ul>

About this Article -

This article was crafted using a human-AI hybrid and collaborative approach. AI assisted our team with initial drafting, research insights, identifying key questions, and image generation. Our human editors guided topic selection, defined the angle, structured the content, ensured factual accuracy and relevance, refined the tone, and conducted thorough editing to deliver helpful, high-quality information.See our About page for more information.

This article is based on research published under:

DOI-LINK: 10.3906/biy-1511-93, Alternate LINK

Title: Stably Expressed Reference Genes During Differentiation Ofbone Marrow-Derived Mesenchymal Stromal Cells

Subject: Cell Biology

Journal: TURKISH JOURNAL OF BIOLOGY

Publisher: The Scientific and Technological Research Council of Turkey (TUBITAK-ULAKBIM) - DIGITAL COMMONS JOURNALS

Authors: Ilgın Cagnan, Fatima Aerts Kaya, Duygu Uçkan, Ayşen Günel Özcan

Published: 2017-01-01

Everything You Need To Know

Why are mesenchymal stromal cells, or MSCs, important in regenerative medicine?

Mesenchymal stromal cells, or MSCs, are valuable because they can differentiate into various cell types like bone, cartilage, and fat, making them useful in regenerative medicine for treating injuries and diseases. The varying characteristics of MSCs depending on their source (like bone marrow or adipose tissue) and lab conditions highlight the need for careful experimental design to ensure reliable results. Understanding these variables is essential for harnessing the full potential of MSCs in therapeutic applications.

Why is it important to use reference genes in quantitative polymerase chain reaction, or qPCR, when studying MSCs?

Quantitative polymerase chain reaction, qPCR, is used to measure gene expression in MSCs, but its accuracy depends on the stability of reference genes. These reference genes act as controls to normalize data, but their expression can fluctuate, leading to misleading results. Identifying stable reference genes is crucial for reliable qPCR analysis and accurate interpretation of MSC behavior.

How did the researchers identify stable reference genes for bone marrow-derived MSCs differentiating into fat and bone cells?

The research evaluated nineteen potential reference genes in bone marrow-derived MSCs, or BM-MSCs, differentiating into adipocytes (fat cells) and osteoblasts (bone cells). These genes were assessed using NormFinder and GeNorm software to determine their expression stability. The goal was to identify genes with consistent expression levels regardless of the differentiation stage. This meticulous evaluation is essential to pinpoint genes that ensure accurate data normalization in qPCR experiments.

Which reference genes were found to be the most stable in bone marrow-derived MSCs differentiating into adipocytes and osteoblasts, and why was RPLP0 recommended?

The study found that PBGD was the most stable gene when analyzing undifferentiated MSCs, adipocytes, and osteoblasts together, closely followed by G6PDH. However, due to low expression levels, RPLP0 is a more suitable option because of its higher expression with reasonably good stability. Choosing the right reference gene depends on the experimental setup to ensure accurate and reliable results.

What are the implications of using unstable reference genes in qPCR experiments involving bone marrow-derived MSCs?

Using unstable reference genes in qPCR experiments with bone marrow-derived MSCs can lead to inaccurate results and misinterpretations of MSC behavior. This can affect the conclusions drawn from the research and the potential applications of MSCs in regenerative medicine. Selecting appropriate reference genes is essential for ensuring the reliability and validity of experimental findings.