RNA Analysis

RNA is single stranded, forming complex secondary structures through intra-molecular base pairing.

AATI RNA Analysis Photo

What is RNA?

The research record of RNA is rather short. It was not until the first half of the twentieth century that RNA was identified as a biological macromolecule independent of DNA by enterprising carbohydrate chemists. Since that distinction was made, multiple classes of RNA have been found, including: messenger RNA (mRNA), ribosomal RNA (rRNA), small RNA, microRNA, and long non-coding RNA. Similar to DNA, RNA poses a unique set of challenges to researchers, including susceptibility to ever-present RNases, degradation, and shearing from pipetting.

The molecular cousin of DNA, RNA plays myriad significant roles in molecular biology, including: serving as a genome, enzymatic activity, and participating in every step of gene expression. Due to the ubiquity of RNA in biology, scientists have invested time and effort into understanding its chemical and biological properties.

In the endeavor to understand RNA biology, researchers have developed a multitude of relevant molecular biology techniques, ranging from reverse transcription PCR and agarose gel electrophoresis, to microarrays and next-generation sequencing. In the interest of streamlining and simplifying RNA analysis and QC workflows, Advanced Analytical Technologies, Inc. produces several solutions for RNA workflows.

RNA is important to numerous molecular biology applications, including: qPCR, NGS, and microarrays. An in-depth understanding of RNA biology impacts a wide range of biological research, from agriculture and medicine to evolution and genome editing.

Ubiquitous in biology, RNA is crucial to diverse and significant processes in molecular biology. As such, it is a biological macromolecule of interest to scientists of diverse backgrounds and interests. Pivotal to the understanding of everything from the origin of life to cancer biology, numerous applications have been developed to better understand the biological roles of RNA. Sophisticated downstream applications, notably NGS, require the use of RNA of a suitable quality to ensure reliable results. However, traditional methods of RNA analysis lack the throughput, sensitivity, accuracy required to confidently assess the suitability of RNA for downstream applications. For example, RNA agarose gel electrophoresis is a low-throughput solution that, in addition to lacking suitable resolution and quantification capabilities, is tedious and time-consuming.

Total RNA

Ascertaining the quality of total RNA is of absolute importance to microarray analysis, NGS, and other applications sensitive to RNA degradation and contamination. Legacy methods of RNA analysis demand high input concentrations with limited resolution and quantification capabilities. The Fragment Analyzer™, the Fragment Analyzer INFINITY®, and the FEMTO Pulse™ provide superior quantification, qualification, and sizing of total RNA samples.

To aid in the empirical assessment of total RNA quality, AATI developed the RNA Quality Number (RQN) for PROSize® Data Analysis Software. Automatically calculated by PROSize for every total RNA sample, the RQN accounts for several factors including the ratio between ribosomal peaks, the small RNA region, and resolution between peaks. Reported on a scale of 1 to 10, higher values indicate a higher quality total RNA sample.

The Fragment Analyzer and the Fragment Analyzer INFINITY have two reagent kits for the consistent assessment of total RNA quality, quantity, and size: the DNF-471 Standard Sensitivity RNA Analysis Kit and the DNF-472 High Sensitivity RNA Analysis Kit. The separation resolutions of these kits provide researchers with a clear look at the diverse separation profiles of total RNA (Download Application Note: "Plant RNA Degradation Detection with the Fragment Analyzer").

Figure 1. Separation of total RNA extracted from rice root. The purple and pink peaks represent the large and small ribosomal RNA peaks, respectively. The small RNA region is comprised of the peaks and smears below 200 nt. Capillary electrophoresis was performed using the DNF-471 Standard Sensitivity RNA Analysis Kit on a Fragment Analyzer Automated CE System equipped with a Short Capillary Array (33-55).

The Fragment Analyzer is adaptable to low or high throughput facilities interested in total RNA quality, separating as few as 12 samples to as many as 288 samples. However, for truly high throughput environments, such as genomics laboratories, the Fragment Analyzer INFINITY offers the best option for total RNA analysis with either the DNF-471 Standard Sensitivity RNA Analysis Kit or the DNF-472 High Sensitivity RNA Analysis Kit.

If sensitivity is a primary concern, consider the FP-1201 Ultra-Sensitivity RNA Analysis Kit. Conserve precious sample and properly account for low concentration transcripts within a total RNA sample. The RQN is fully compatible with the FP-1201 Ultra-Sensitivity RNA Analysis Kit, enabling empirical quality analysis of low concentration total RNA samples.

Messenger RNA

Messenger RNA (mRNA), the intermediary between DNA and protein, is of interest to researchers as it provides insight into gene activity. The large-scale sequencing of mRNA from an organism – termed transcriptomics – enables researchers to profile what genes and genomic regions are active under different conditions. As with other NGS applications, the proper QC analysis of raw nucleic acids ensures only suitable samples are sequenced.

Figure 2. Messenger RNA (mRNA) sample extracted and purified from rat kidney tissue. Capillary electrophoresis was performed with the DNF-472 High Sensitivity RNA Analysis Kit using the mRNA separation method on a Fragment Analyzer Automated CE System equipped with a Short Capillary Array (33-55).

Figure 3. Low concentration (30 pg/µL) separation of messenger RNA (mRNA). Capillary electrophoresis was performed using the FP-1201 Ultra-Sensitivity RNA Analysis Kit on a FEMTO Pulse Automated Pulsed-Field CE Instrument.

Small RNA

Small RNA is a broad and growing classification, including: microRNA, siRNA, piRNA, and snRNA. Analysis of small RNAs is often difficult as many methods of analysis lack the minimum resolution and sensitivity required for accurate separation, sizing, and quantification. The DNF-470 Small RNA Analysis Kit for the Fragment Analyzer and the Fragment Analyzer INFINITY brings supreme sensitivity, variable throughput, and remarkable resolution to small RNA QC analysis.

Figure 4. Total RNA sample extracted from rice root and separated using the DNF-470 Small RNA Analysis Kit. Capillary electrophoresis was performed with a Fragment Analyzer Automated CE System equipped with a Short Capillary Array (33-55). The red lines on the electropherogram indicate the different regions of the sample: the microRNA Region (10 nt – 40 nt) and the Small RNA Region (10 nt – 200 nt).

FFPE RNA

Reliable QC analysis of FFPE RNA is essential for confident NGS results. FFPE RNA samples exhibit higher levels of degradation and crosslinking, limiting the application of the RQN to these samples. The DV200 quality metric for PROSize was developed by scientists at Illumina to address the unique QC challenges of FFPE RNA. The DV200 provides researchers with a powerful tool to reliably parse FFPE RNA samples suitable for NGS from unsuitable samples, conserving time and costs.

RNA Analysis Kits

Several instrument offerings from Advanced Analytical Technologies, Inc. (AATI) that provide effective solutions for the QC analysis of RNA: the Fragment Analyzer Automated CE System, the Fragment Analyzer INFINITY Automated CE System, and the FEMTO Pulse Automated Pulsed-Field CE Instrument. In conjunction with the relevant reagent kits, the instruments from AATI provide unmatched sizing and quantification capabilities.

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