Quality Assessment of Total RNA Using MultiNA Automated Analysis

User Benefits

- MultiNA automates almost the entire electrophoresis workflow. - MultiNA automatically determines the ratio of 28S rRNA to 18S rRNA (28S/18S) and displays it in a report. - MultiNA outputs numeral results that allow an objective evaluation of the total RNA quality.

Introduction

The extraction of total RNA from biological tissue samples is an important process and the starting point for a wide range of analytical techniques, such as northern blotting, cDNA library construction, cloning, sequencing (next-generation sequencing and Sanger sequencing), and PCR (real-time PCR and qualitative PCR). Sample collection, preparation, and storage conditions also have a major impact on the quality and yield of a total RNA sample. Following extraction, the quality of total RNA in the sample is assessed by ultraviolet (UV) spectroscopy and electrophoresis (denatured agarose gel electrophoresis). The ratio of sample absorbance at 260 to 280 nm is used to test RNA sample purity, where a ratio (260/280 nm) of 2.0 is generally considered the benchmark for pure RNA. A ratio below 2.0 raises concerns over adulteration of the total RNA sample by proteins or reagents used in sample preparation. RNA is also considered more susceptible to degradation than DNA. Electrophoresis can be used to assess the degree of degradation of total RNA samples based on the above- mentioned ratio of 28S rRNA to 18S rRNA. When analyzed by electrophoresis, a sample free from impurities displays a clear 28S rRNA peak and 18S rRNA peak. Since the size ratio of 28S rRNA to 18S rRNA in vivo is 2:1, the 28S and 18S peaks in a high- quality RNA sample are detected in the ratio of 2:1 after electrophoresis analysis. When electrophoresis analysis of RNA is performed on denatured agarose gel, every step of the process is labor intensive, from gel preparation and sample application to performing electrophoresis, signal detection, and clean up. This article describes a quality assessment of a total RNA sample performed using a Shimadzu microchip electrophoresis system.

23 de mayo de 2023 GMT