Comparative Evaluation of RNAlater Solution and Snap Frozen Methods for Gene Expression Studies in Different Tissues

Huseyin Ozkan,Enes Kerman

doi:10.2478/rrlm-2020-0024

Abstract

Abstract Introduction: Freezing of tissues with liquid nitrogen is the most common method in studies performed at the RNA level. However, the use of RNA stabilization solutions has become a popular alternative method. The aim of this study is to investigate the effectiveness of RNAlater on RNA stabilization in different tissues. Material and Methods: In this study, RNA were isolated from the lung, heart, liver and skeletal muscle tissues of rats that were frozen with liquid nitrogen (snap frozen, SF group) or stored in RNAlater solution (RL group), and the changes in concentration, purity, reference genes expression, and fold-change levels between groups were analyzed. Results: In the RL group, the concentration of RNA isolated from the liver tissues was higher (P<0.05), whereas the A260/280 ratio was lower in the heart and liver tissues (P<0.05). PPIA and SRP72 genes were found to have lower Ct values in the heart tissues of rats in the RL group (P<0.05 and P<0.001, respectively) than the SF group. Expression levels of PPIA, ACTB, and SRP72 genes across the tissues were found to be different between the groups (P<0.05). The gene expression level examined in terms of fold-change was significantly different in the RL group (upregulated up to 4 folds and downregulated about 0.5 fold) (P< 0.05). Conclusions: The results showed that RNAlater can maintain the RNA integrity and can also change the results of gene expression because it does not inhibit biological activity. The snap freezing method is more reliable because gene expression is more stable in tissues frozen with liquid nitrogen.

Highlights

Freezing of tissues with liquid nitrogen is the most common method in studies performed at the RNA level
Possible RNA quality loss that may occur in the lung, heart, liver, and skeletal muscle tissues stored in RNAlater was studied in terms of Ct and gene expression through electrophoretic and spectrophotometric controls as well as Peptidylprolyl Isomerase A RL (PPIA) (Peptidylprolyl Isomerase A), Beta Actin GAPDH (ACTB) (Beta Actin), GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase), and SRP72 (Signal Recognition Particle 72) genes, which are commonly used as reference genes [17,18,19,20,21]
The concentration and purity values of total RNA isolated from the lung, heart, liver, and skeletal muscle tissues in the Snap Frozen SRP72 (SF) and RL groups were determined to be appropriate for cDNA and qPCR applications

Summary

Introduction

Freezing of tissues with liquid nitrogen is the most common method in studies performed at the RNA level. Material and Methods: In this study, RNA were isolated from the lung, heart, liver and skeletal muscle tissues of rats that were frozen with liquid nitrogen (snap frozen, SF group) or stored in RNAlater solution (RL group), and the changes in concentration, purity, reference genes expression, and fold-change levels between groups were analyzed. Possible RNA quality loss that may occur in the lung, heart, liver, and skeletal muscle tissues stored in RNAlater was studied in terms of Ct and gene expression through electrophoretic and spectrophotometric controls as well as PPIA (Peptidylprolyl Isomerase A), ACTB (Beta Actin), GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase), and SRP72 (Signal Recognition Particle 72) genes, which are commonly used as reference genes [17,18,19,20,21]

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Discussion

Conclusion