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RNA Sample Quality Control Analysis

RNA Analysis with the Agilent Automated Electrophoresis portfolio

What is RNA?

Ribonucleic acid, RNA for short, is single stranded nucleic acid and one of the major biological macromolecules, essential for all forms of life on earth, along with DNA and protein. The main role of RNA is to act as a messenger to carry instructions from DNA in the form of amino acid sequences to ribosomes for protein synthesis. As proteins are synthesized, they have many higher-order responsibilities, including acting as enzymes and structural components, and playing different roles in cellular signaling processes.

Thus, by being the messenger for protein synthesis, RNA is critical to gene expression and in the regulation of cellular processes such as cell division and cell growth. Because of these crucial roles, RNA is a topic of interest to scientists seeking a deeper understanding of biology, and several applications have developed which require the use of high-quality RNA. More recent technologies are enabling the use of lower quality samples, such as formalin-fixed paraffin-embedded (FFPE).

Since RNA can be found in many different lengths and shapes and can be easily degraded during extraction, quality control (QC) steps are essential.

The Agilent Automated Electrophoresis portfolio is comprised of several instruments, including the Bioanalyzer, Fragment Analyzer, TapeStation, and Femto Pulse systems, each offering different advantages such as throughput, sensitivity, speed, and resolution. Together, the Automated Electrophoresis instruments provide superior quantification, qualification, and sizing of RNA to allow for confident assessment of samples to be used for downstream applications.

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. Agilent systems provide superior quantification, qualification, and sizing of total RNA samples.

Messenger RNA

The large-scale sequencing of mRNA from an organism enables researchers to profile what genes and genomic regions are active under different conditions. Agilent systems provide reliable QC analysis of mRNA and ribo-depleted total RNA.

Small RNA

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. An Agilent system brings supreme sensitivity, variable throughput, and remarkable resolution to small RNA QC analysis.

FFPE RNA

Reliable QC analysis of FFPE RNA is essential for confident NGS results. Agilent systems utilize a specalized quality metrics to address the unique QC challenges of FFPE RNA.

RNA Analysis Kits

Several instrument offerings from Agilent provide effective solutions for the QC analysis of RNA. With the relevant reagent kits, the instruments provide unmatched sizing and quantification capabilities.

RNA Application Notes

Don't stop learning here, read more about RNA analysis in our application notes.

For Research Use Only. Not for use in diagnostic procedures.

Total RNA Qualification

To aid in the empirical assessment of total RNA, each system features a quality metric for objective assessment of RNA quality: the RNA Integrity Number (RIN) from the Bioanalyzer system, RNA Quality Number (RQN) from the Fragment Analyzer and Femto Pulse systems, and RNA Integrity Number Equivalent (RINe) from the TapeStation systems. Each system automatically calculates a quality score for every sample, taking into account the ratio between ribosomatl peaks, small RNA region, and resolution between peaks. The metrics are reported on a scale of 1 to 10, with the higher values indicating a higher-quality total RNA sample.

2100 Bioanalyzer system

The 2100 Bioanalyzer system offers two kits for accurate QC of total RNA:

Both kits have a specific assay for prokaryotic and eukaryotic RNA samples and plant RNA. The automatic evaluation of the unambiguous RIN, calculation of ribosomal ratios, and quantitative data offered by these assays has made the 2100 Bioanalyzer a well-established standard for RNA QC.

Separation of total RNA extracted from human lymph tissue. The pink and green lines designate the 18S and 28S ribosomal RNA peaks, respectively. The small fragments that appear before the ribosomal peaks are the indicative of the small RNA region. Electrophoresis was performed using the RNA 6000 Nano kit on a 2100 Bioanalyzer system. This sample has a RIN of 9.8, indicating high-quality RNA.

Total RNA sample on the Bioanalyzer

Fragment Analyzer systems

The Fragment Analyzer systems have two reagent kits for the consistent assessment of total RNA quality, quantity, and size:

ProSize data analysis software automatically calculates a quantitative quality score, the RQN, for each sample. The excellent resolution of these kits provides researchers a clear look at the unique profiles of total RNA, separate from small and degraded RNA, increasing the accuracy of the RQN score.

Universal Mouse Reference on the Fragment Analyzer

Separation of Universal Mouse Reference total RNA. The purple and pink peaks represent the large and small ribosomal RNA peaks, respectively. The small fragments seen below 200 nt is indicative of the small RNA region. Capillary electrophoresis was performed using the RNA kit (15 nt) on a Fragment Analyzer system equipped with a short capillary array, 33 cm. The RNA Property Summary (inset) includes the sample concentration, ribosomal ratio, and the RNA Quality Number (RQN). This sample has an RQN of 9.9, indicating high-quality RNA.

TapeStation systems

The TapeStation systems offer two assays for efficient and reliable RNA characterization, quality assessment, and quantification:

The RINe is directly comparable to the RIN of the Bioanalyzer system and delivers fast and objective evaluations of RNA integrity for both eukaryotic and prokaryotic total RNA samples.

Four rat total RNA samples (300 ng/μL) with different levels of RNA degradation were analyzed on a TapeStation system using the RNA ScreenTape assay. The determined RINe values are shown under the gel image. A lower RINe indicates a more degraded sample, while a larger RINe indicates high-quality RNA.

Rat Total RNA Samples on the TapeStation

Femto Pulse system

If sensitivity is a primary concern, the Ultra Sensitivity RNA kit (p/n FP-1201) conserves precious sample, while properly accounting for low concentration transcripts within a total RNA sample. The ProSize data analysis software and the RQN quality metric are fully compatible with the Femto Pulse, enabling advanced quality analysis of low concentration total RNA samples.

Universal Mouse Reference on the Femto Pulse

Separation of Universal Mouse Reference total RNA. The purple and pink peaks represent the large and small ribosomal RNA peaks, respectively. Capillary electrophoresis was performed using the US RNA kit on a Femto Pulse system. The RNA Property Summary (inset) includes the sample concentration, ribosomal ratio, and the RNA Quality Number (RQN). This sample has an RQN of 10.0, indicating high-quality RNA.

For Research Use Only. Not for use in diagnostic procedures.

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 enables researchers to profile what genes and genomic regions are active. In a typical workflow involving mRNA, total RNA is first analyzed for sample integrity before ribosomal RNA (rRNA) depletion, to ensure that the RNA is not degraded. The mRNA is then analyzed with an mRNA assay to verify that the residual rRNA content is low, as indicated by a low percent rRNA contamination score. As with other NGS applications, the proper QC analysis of nucleic acids ensures that only suitable samples are processed. Quantitative applications for gene expression also rely on having intact mRNA, as degraded total RNA may lead to the absence of some transcripts, and to misinterpretation of the results. Thus, QC steps to ensure a low percentage of rRNA contamination in the mRNA is crucial to the success of these sensitive downstream applications.

Reliable QC analysis of mRNA and ribo-depleted total RNA is achieved with the Agilent Automated Electrophoresis portfolio. For accurate assessment of mRNA, use:

mRNA from Liver Tissue on the Bioanalyzer

2100 Bioanalyzer system

Messenger RNA (mRNA) sample extracted and purified from liver tissue. Electrophoresis was performed with the RNA 6000 Nano kit on a 2100 Bioanalyzer system using the mRNA assay. In this example, the ribosomal contamination is 8.1%.

Fragment Analyzer system

Messenger RNA (mRNA) sample extracted and purified from rat kidney tissue. Capillary electrophoresis was performed with the HS RNA kit using the mRNA separation method on a Fragment Analyzer system equipped with a short capillary array, 33 cm. The mRNA Property Summary indicates the percent of ribosomal contamination present in the sample, in this example 13.6%.

mRNA Sample from Rat Kidney on the Bioanalyzer

mRNA Sample from Rat Kidney on they Femto Pulse

Femto Pulse system

Separation of a mouse kidney mRNA sample at a low concentration (30 pg/µL). Capillary electrophoresis was performed using the Ultra Sensitivity RNA kit on a Femto Pulse system. The mRNA Property Summary indicates the percent of ribosomal contamination present in the sample, in this example 6.8%

In vitro transcribed (IVT) messenger RNA (mRNA)

The use of in vitro transcription (IVT) mRNA is becoming widespread in research areas such as ribozyme and aptamer synthesis, mRNA synthesis, RNA interference, and antisense RNA techniques. While conventional IVT mRNAs are fewer than 100 nucleotides in length, longer RNA transcripts are now being used for gene structure and functional studies. Reliable QC steps are required to produce these synthetic RNAs. Sizing, quantification, and quality assessment of IVT mRNA ranging from 200 to 6,000 nt in size can be obtained using the Fragment Analyzer systems with the RNA kit (15 nt) (p/n DNF-471). For longer transcripts over 6,000 nt, an extended method can be used.

Extended RNA Ladder

An 1,800 nt IVT mRNA sample separated using the RNA kit (15 nt) on a Fragment Analyzer system equipped with a short capillary array, 33 cm.

For Research Use Only. Not for use in diagnostic procedures.

Small RNA

Small RNA is a broad and growing classification, including: microRNA (miRNA), small interfering RNA (siRNA), PIWI-interacting RNA (piRNA), and small nuclear RNA (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 Bioanalyzer and Fragment Analyzer systems are uniquely suited for the supreme sensitivity, variable throughput, and remarkable resolution required for small RNA QC analysis with:

Small RNA sample on the Bioanalyzer

2100 Bioanalyzer system

Total RNA sample extracted from mouse thymus and separated on a 2100 Bioanalyzer system using the Small RNA assay. The area between the green lines represents the Small RNA Region, while the purple dashed lines indicate the microRNA Region (10 to 40 nt). The Region Table (inset) gives the average size, size distribution, and concentration of each region, along with the percentage of the sample that is small RNA and microRNA.

Fragment Analyzer system

Total RNA sample extracted from rice root and separated using the Small RNA kit. Capillary electrophoresis was performed with a Fragment Analyzer system equipped with a short capillary array, 33 cm. The purple lines on the electropherogram indicate the different regions of the sample: the microRNA Region (10 to 40 nt) and the Small RNA Region (10 to 200 nt). The Small RNA Summary table (inset) gives the percentage of the sample that is microRNA, along with the concentrations of the small RNA and microRNA regions.

Small RNA Sample on the Fragment Analyzer

For Research Use Only. Not for use in diagnostic procedures.

FFPE RNA

Reliable QC analysis of FFPE RNA is essential for confident NGS results. FFPE RNA samples exhibit higher levels of degradation and cross-linking, limiting the effectiveness of the RNA quality metrics for these samples. The Automated Electrophoresis systems from Agilent utilize a quality metric, DV200, to address the unique QC challenges of FFPE RNA. The DV200 provides researchers with a tool to reliably classify degraded RNA by size and effectively parse samples suitable for NGS from unsuitable samples, conserving time and costs. Depending on the level of degradation and the DV200 score, researchers can modify their workflows, to still enable the use of FFPE samples in downstream applications. For example, when a specific size is needed and the sample is slightly degraded, the shearing protocol can be adjusted to reach that size. Take advantage of automated evaluation of RNA samples using the DV200 quality metric with any of the Automated Electrophoresis platforms:

RNA Nano Sample on the Bioanalyzer

2100 Bioanalyzer system

FFPE RNA extracted and purified from prostate tissue. Electrophoresis was performed using the DV200 RNA Nano assay on a 2100 Bioanalyzer system. For this sample, the DV200 score is 45%.

Fragment Analyzer system

FFPE RNA sample separated using the HS RNA kit (15 nt) for the Fragment Analyzer systems. Capillary electrophoresis was performed with a short capillary array, 33 cm. The red lines on the electropherogram indicate the DV200 region. For this sample, the DV200 score is 22.6% indicating that a small percentage of the sample is above 200 nt.

FFPE HS RNA sample on the Fragment Analyzer

FFPE RNA sample on the TapeStation

TapeStation system

Comparison of FFPE RNA samples with varying DV200 values and typical electropherogram profiles. Samples were analyzed on a TapeStation system with the RNA ScreenTape assay.

For Research Use Only. Not for use in diagnostic procedures.

RNA Analysis Kits

The Automated Electrophoresis instruments from Agilent provide effective solutions for the QC analysis of RNA: the Bioanalyzer, Fragment Analyzer, TapeStation, and Femto Pulse systems. With the relevant reagent kits, the instruments provide unmatched sizing and quantification capabilities.

Instrument Part Number(s) Reagent Kit Name Sizing Range Qualitative Range Quality Metric
2100 Bioanalyzer system 5067–1511 RNA 6000 Nano kit - 25 - 500 ng/µL RIN, DV200
5067–1513 RNA 6000 Pico kit - 50 - 5,000 ng/µL RIN, DV200
5067–1548 Small RNA kit 6 - 150 nt 50 - 2,000 ng/µL -
Fragment Analyzer systems DNF-470-0275 Small RNA kit 15 – 200 nt 25 – 2,500 pg/µL RQN, DV200
DNF-471-0500
DNF-471-1000
RNA kit (15NT) 200 – 6,000 nt 5 – 500 ng/µL RQN, DV200
DNF-472-0500
DNF-472-1000
HS RNA kit (15NT) 200 - 6,000 nt 50 - 5,000 pg/µL -
TapeStation systems 5067-5576 (ScreenTape)
5067-5577 (Buffer)
5067-5578 (Ladder)
RNA ScreenTape - 25 – 2,500 pg/µL RINe, DV200
5067-5579 (ScreenTape)
5067-5580 (Buffer)
5067-5581 (Ladder)
High Sensitivity RNA ScreenTape - 500 – 10,000 pg/µL RINe, DV200
Femto Pulse system FP-1201-0275 Ultra Sensitivity RNA kit 200 – 6,000 nt 2.5 – 250 pg/µL RQN

For Research Use Only. Not for use in diagnostic procedures.

RNA Application Notes

Quality Analysis of Eukaryotic Total RNA with the Agilent 5200 Fragment Analyzer System

The Agilent 5200 Fragment Analyzer and associated RNA Kits allow for easy analysis of total RNA quality and concentration.

Plant RNA Degradation Detection Using the Agilent 5200 Fragment Analyzer System

The Agilent Fragment Analyzer and associated RNA Kits allow for easy analysis of RNA quality in RT-PCR, microarray analysis, and next-generation sequencing.

Monitoring Library Preparation for Next-Generation Sequencing in systems Biology Omics Analysis

Evaluation of the Agilent 4150 TapeStation system with ScreenTape assay to monitor exome and transcriptome library preparation for systems biology omics analysis.

Comparison of RIN and RINe Algorithms for the Agilent 2100 Bioanalyzer and the Agilent 2200 TapeStation systems

This Technical Overview demonstrates that RINe values obtained from the Agilent 2200 TapeStation system

Comparison of RIN and RQN for the Agilent Bioanalyzer and the Fragment Analyzer Systems

This technical overview examines the RIN from the Bioanalyzer and the RQN from the Fragment Analyzer. Comparison of the RIN and RQN is performed with several eukaryotic and prokaryotic RNA samples that have various degrees of degradation.

Assessing Integrity of Insect RNA

This App Note shows the applicability of total RNA quality assessment of different insect species with the 2100 Bioanalyzer System.

Analysis of miRNA content in total RNA preparations using the Agilent 2100 Bioanalyzer

Advances in miRNA research have led to the increased demand for techniques appropriate for the analysis of this small RNA subclass.

For Research Use Only. Not for use in diagnostic procedures.