A 5S

5.8S

miRNA

Small RNA

Improved Analysis of RNA, FFPE RNA and microRNA using the Fragment Analyzer™

Kit-Sum Wong, Alec Boeckholt, Jon Hagopian, Jeremy Kenseth, Steve Siembieda

Advanced Analytical Technologies, Inc., Ames, IA 50010

Introduction

Next Gen Sequencing (NGS) of RNA is growing quickly and may soon be the method of choice to analyze RNA sequence composition, expression

levels, splice sites and variations. Inherent to its analysis, by any molecular method, is an understanding of both the quality and quantity of this

extracted nucleic acid. Today, high quality RNA can be easily extracted from a few to many thousand freshly prepared cells. New technologies such

as the Fluidigm C1™ prep system can be used to isolate, extract and convert RNA from single cells into sequenceable libraries.

For poor quality RNA samples, like formalin-fixed paraffin-embedded (FFPE) or poorly frozen tissues, RNA quality can suffer considerably. Though

sequenceable RNA can be obtained and converted into a library preparation, care must be taken to understand the initial RNA quality in order to

generate reliable results. Additionally, microRNA analysis by NGS sequencing is gaining traction; however, instruments capable of analyzing the

quality of these RNA species are limited.

Tested and confirmed to analyze all types of RNA, the Fragment Analyzer™ offers both hardware and software features that can improve laboratory

workflow by streamlining experimental setup, analyzing samples in parallel and providing intuitive customizable data analysis. For total RNA quality,

the Fragment Analyzer™ RNA Quality Number (RQN) has been validated across a variety of sample types and quality to be equivalent to the

traditional integrity score value. For FFPE RNA, recent methods developed by both Illumina and Advanced Analytical show how a DV200 metric (i.e.

the percentage of RNA >200 nucleotides) can be a better predictor of sequencing outcome than the traditional integrity score value. In the case of

microRNA, the Fragment Analyzer™ can accurately assess both the quality and quantity of these RNA species in a single separation with high

resolution and sensitivity. For single cell transcriptomics, the Fragment Analyzer™ is an ideal tool for verifying single cell capture and amplification

when incorporated into the C1™ sequencing workflow. Data regarding these applications will be presented.

Samples and Reagents

The mRNA and total RNA and samples were from purchased from Clontech. The FFPE RNA samples and data were obtained from Illumina

(courtesy of Lisa Watson). The small RNA samples were from the Scripps Research Institute (courtesy of Tony Mondala). The Fluidigm C1™ cDNA

sample products and data were obtained from Stephen Quake’s Laboratory at Stanford University (courtesy of Norma Neff and Gary Mantalas).

Instrumentation and Methods

Parallel Capillary Electrophoresis was performed on the Fragment Analyzer™ platform, in either the 96-capillary or 12-capillary array format. A high

output LED source provided an excitation wavelength of 470 nm with emission collected at 500-600 nm. The capillary array was conditioned with

Capillary Conditioning Solution, and then filled with the appropriate Separation Gel. For each run, a pre-loaded Separation Method was queued as

outlined in the respective gel kit user manual. Data analysis was performed using PROSize® 2.0 software (Advanced Analytical).

• 96-capillary or 12-capillary capability

• 96 or 12 individual CGE separations are performed in parallel simultaneously

• Fully automated workflow enables continuous loading/exchange of up to three 96-well plates along with method scheduling while the instrument

is running

• Measures both quantity (concentration) and quality (size/distribution) of DNA fragments, genomic DNA, NGS library smears, total RNA,

messenger RNA, or microRNA

Advanced Analytical Technologies Fragment Analyzer™

Analysis of Total RNA – RNA Quality Number (RQN)

Comparison of small RNA Analysis by Different Methods

Experimental

• The mRNA was analyzed in a Fragment Analyzer™ using the DNF-491 High Sensitivity RNA Analysis Kit

• Software reports size, concentration and % rRNA contamination

Figure 2 Rat liver total RNA

Comparison of Total RNA Analysis by Different Methods

Expected

Concentrations

Spectrophotometry

(UV-Vis) Fluorometry

Chip-Based

Electrophoresis Fragment Analyzer ™

Conc. (ng/µL) Conc. (ng/µL) Conc. (ng/µL) Conc. (ng/µL) Conc. (ng/µL)

Avg %CV Avg %CV Avg %CV Avg %CV

500 533.0 1.2 541.0 6.2 541.0 1.0 561.4 1.7

250 282.7 0.4 255.7 6.4 323.0 0.9 256.8 9.7

100 111.4 1.0 110.7 13.3 141.0 4.0 93.2 4.3

25 27.7 2.9 24.1 0.9 40.0 3.5 22.1 0.7

5 4.9 6.6 4.5 2.1 13.0 32.6 5.6 29.7

The total RNA samples were analyzed in a Fragment Analyzer™ using the DNF-471 Standard Sensitivity RNA Analysis Kit (15nt) (A). The

same samples were analyzed by UV spectrophotometry, fluorometry and chip-based electrophoresis (B).

A B

Analysis of microRNA and small RNA

• RNA sample analyzed with the Fragment Analyzer™ using the DNF-470 Small RNA Analysis Kit

• Software enables input of Small RNA Size Regions, and outputs % microRNA, small RNA and micro RNA concentrations

• Small RNA sample analyzed by the Fragment Analyzer™ using the DNF-470 Small RNA Analysis Kit (A) or by chip-based electrophoresis (B)

• Fragment Analyzer™ yields similar profile with improved resolution of lower marker from miRNA region

Methods Small RNA Concentration, pg/µL

10nt - 150nt

miRNA Concentration, pg/µL

10nt – 40nt miRNA/small RNA ratio, %

Fragment Analyzer™ 1,556 630 40.5

Chip-Based Electrophoresis 1,980 695 35

Analysis of FFPE RNA Samples and Calculation of DV200 Metric

Example FFPE RNA sample result and calculated DV200 value analyzed by the Fragment Analyzer™ system, using the DNF-471 Standard

Sensitivity RNA Analysis Kit (15 nt).

Fragment Analyzer™ QC of Fluidigm C1™ Transcriptome (mRNA Seq) cDNA

• The Fluidigm C1™ Single-Cell Auto Prep System is an innovative microfluidic technology enabling the capture, processing and profiling of

single cells for genomic analysis

• The Fragment Analyzer™ is an ideal complementary QC tool, capable of analyzing 96-well plate formatted cDNA product for presence, size,

and concentration following single cell capture, lysis, RT and amplification prior to library preparation. Analysis is performed using the DNF-

474 High Sensitivity NGS Fragment Analysis kit.

• The Fragment Analyzer™ provides a sensitive, automated solution to measure quantity and quality of RNA

from a variety of sources

• A variety of application kits and associated software tools have been developed for efficient analysis of total

RNA, messenger RNA, and small/microRNA

• In addition to RNA analysis, the Fragment Analyzer™ is also used for DNA fragment, NGS library, and gDNA

analysis applications

Analysis of mRNA

Results

Summary

28S/18S = 1.1

100 ng/uL Human Heart total RNA

100 ng/uL Human Heart total RNA + 10 ng/uL Human gDNA

• The total RNA sample was analyzed in a Fragment Analyzer™ using the DNF-471 High Sensitivity RNA Analysis Kit (15nt)

• Software reports concentration, size, 28S/18S rRNA ratio, and RQN

5S

5.8S

miRNA

Small RNA

B

5.8S

5S

miRNA

Small RNA

• Total RNA sample spiked with human gDNA

• Fragment Analyzer™ resolves and detects presence of gDNA

• The Fragment Analyzer™ system provides very good quantitative agreement to other commonly employed methods for total RNA analysis

• Fragment Analyzer™ RQN is based on proprietary algorithm

• RQN provides excellent correlation to RIN as validated by comparison

to over 100 sample of various origin, concentration and purity

The accurate assessment of RNA quality is critical for successful RNA sequencing. This is true especially for RNA sequencing of FFPE samples,

where substantial differences in quality may exist between different specimens due to storage conditions or fixation methods.

Illumina scientists have determined that the percentage of RNA fragments > 200 nt in a sample (termed the “DV200” value) is a more reliable

predictor of RNA quality when analyzing highly degraded RNA sample quality, compared to the traditional integrity score or value.1 Using the DV200

as a guide, recommended minimal RNA input amounts have been determined for ensuring reliable, robust sequencing results.

The can be automatically calculated by the Fragment Analyzer™ system, using a customized DV200 separation and data analysis method file.

% Total (Range 200 nt – 20000 nt) = DV200

1Evaluating RNA Quality from FFPE Samples. Illumina Technical Note, Pub. No. 470-2014-001. Available for download at:

http://support.illumina.com/sequencing/sequencing_kits/truseq_rna_access_library_prep_kit/questions.ilmn

“No Cell” Result: Peaks are ERCC RNA internal spike

control amplicons (Ambion, NIST-ABMS)

Positive Result: cDNA distribution average size and

sample concentration determined with smear analysis