Improved Analysis of RNA, FFPE RNA and microRNA using the

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
Analysis of Total RNA – RNA Quality Number (RQN)
Introduction
Analysis of microRNA and small RNA
Small RNA
Next Gen Sequencing (NGS) of RNA is growing quickly and may soon be the method of choice to analyze RNA sequence composition, expression
miRNA
levels, splice sites and variations. Inherent to its analysis, by any molecular method, is an understanding of both the quality and quantity of this
5S
extracted nucleic acid. Today, high quality RNA can be easily extracted from a few to many thousand freshly prepared cells. New technologies such
5.8S
Figure 2 Rat liver total RNA
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
• The total RNA sample was analyzed in a Fragment Analyzer™ using the DNF-471 High Sensitivity RNA Analysis Kit (15nt)
workflow by streamlining experimental setup, analyzing samples in parallel and providing intuitive customizable data analysis. For total RNA quality,
• Software reports concentration, size, 28S/18S rRNA ratio, and RQN
the Fragment Analyzer™ RNA Quality Number (RQN) has been validated across a variety of sample types and quality to be equivalent to the
100 ng/uL Human Heart total RNA
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.
Experimental
• 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
100 ng/uL Human Heart total RNA + 10 ng/uL Human gDNA
28S/18S = 1.1
Comparison of small RNA Analysis by Different Methods
Samples and Reagents
Small RNA
The mRNA and total RNA and samples were from purchased from Clontech. The FFPE RNA samples and data were obtained from Illumina
A
(courtesy of Lisa Watson). The small RNA samples were from the Scripps Research Institute (courtesy of Tony Mondala). The Fluidigm C1™ cDNA
miRNA
Small RNA
5S
B
5S
sample products and data were obtained from Stephen Quake’s Laboratory at Stanford University (courtesy of Norma Neff and Gary Mantalas).
Instrumentation and Methods
• Fragment Analyzer™ resolves and detects presence of gDNA
5.8S
5.8S
• Fragment Analyzer™ RQN is based on proprietary algorithm
• Total RNA sample spiked with human gDNA
• RQN provides excellent correlation to RIN as validated by comparison
to over 100 sample of various origin, concentration and purity
miRNA
Parallel Capillary Electrophoresis was performed on the Fragment Analyzer™ platform, in either the 96-capillary or 12-capillary array format. A high
Comparison of Total RNA Analysis by Different Methods
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).
A
• Small RNA sample analyzed by the Fragment Analyzer™ using the DNF-470 Small RNA Analysis Kit (A) or by chip-based electrophoresis (B)
B
• Fragment Analyzer™ yields similar profile with improved resolution of lower marker from miRNA region
Advanced Analytical Technologies Fragment Analyzer™
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
Methods
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).
• 96-capillary or 12-capillary capability
• 96 or 12 individual CGE separations are performed in parallel simultaneously
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
Fragment Analyzer™ QC of Fluidigm C1™ Transcriptome (mRNA Seq) cDNA
• Fully automated workflow enables continuous loading/exchange of up to three 96-well plates along with method scheduling while the instrument
• The Fragment Analyzer™ system provides very good quantitative agreement to other commonly employed methods for total RNA analysis
is running
• Measures both quantity (concentration) and quality (size/distribution) of DNA fragments, genomic DNA, NGS library smears, total RNA,
messenger RNA, or microRNA
Analysis of FFPE RNA Samples and Calculation of DV200 Metric
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.
Results
Analysis of mRNA
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.
“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
• 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 DNF474 High Sensitivity NGS Fragment Analysis kit.
Summary
% Total (Range 200 nt – 20000 nt) = DV200
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).
• The mRNA was analyzed in a Fragment Analyzer™ using the DNF-491 High Sensitivity RNA Analysis Kit
• Software reports size, concentration and % rRNA contamination
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
• 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

Download Report