Technical Note: RNA Sequencing

Evaluating RNA Quality from FFPE Samples
Guidelines for obtaining high-quality RNA sequencing results from degraded RNA withIllumina RNA enrichment assays
Introduction
Formalin-fixed paraffin-embedded (FFPE) archival tissue samples
provide an invaluable repository of information for disease research,especially cancer. However, FFPE samples can be difficult to process
in many molecular biology assays because the fixation process
and subsequent tissue storage methods often cause nucleic aciddegradation, resulting in fragmented RNA transcripts.1,2 Illumina RNA
enrichment assays address these challenges by capturing the coding
regions directly, rather than by poly(A) capture or amplification by sitespecific primers (Figure 1).
Illumina offers 3 RNA enrichment assays that are compatible withFFPE tissues. The TruSeq® RNA Access Library Preparation Kit
focuses on coding regions from the whole transcriptome, enabling
RNA sequencing (RNA-Seq) with less input RNA and fewer reads,
and increasing the number of samples per run for more cost-effective
transcriptome analysis. The TruSight® RNA Pan-Cancer Panelassesses 1,385 cancer-associated genes, providing a comprehensive
solution for RNA expression and fusion discovery in FFPE samples.
The TruSight® RNA Fusion Panel is designed more specifically toassess 507 cancer-associated fusion genes associated with cancer,and enables detection of novel fusions.

A. Pool stranded RNA-Seq libraries

Biotin probes

B. Hybridize biotinylated probes to targeted regions

Obtaining reliable, reproducible results from degraded RNA samples
is possible if the RNA quality is accurately assessed. Effectively
evaluating RNA quality is a critical step in successful RNA sequencing.
This technical note provides guidance for accurately evaluating RNA
samples to obtain high-quality RNA-Seq results.

Streptavidin beads

Variability of FFPE Samples
The quality of RNA isolated from FFPE samples can vary widely
among different specimens, or within different samples from thesame specimen (Figure 2). RNA undergoes substantial chemical
modification during formalin fixation: nucleic acids are crosslinked
to proteins and RNA transcripts are degraded to smaller fragments.
Differences in formalin fixation methods and age of archival tissue
samples add further variation to RNA quality.1-2 RNA enrichment library
prep kits have been optimized to ensure high-quality RNA sequencing
data from degraded FFPE samples and to allow comparison acrosssamples that vary in quality. These kits allow researchers to take fulladvantage of the high reproducibility and broad dynamic range ofIllumina NGS sequencing. But it is important to evaluate the qualityof each FFPE sample before proceeding with library preparation.
Although most FFPE samples perform well with the RNA enrichment
probe sets, some highly degraded samples contain RNA fragments
smaller than the optimal size range for efficient target-capture andlibrary preparation.

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

C. Capture using streptavidin beads

D. Elution from beads
Figure 1: RNA Enrichment Chemistry—Illumina RNA enrichment assays
provide a simple and streamlined method for isolating targeted regions of interest
from challenging RNA samples.

Technical Note: RNA Sequencing
Table 1: RIN and DV200 Values From FFPE Samples
SampleRIN

DV200*

Breast Normal

2.3

77

Breast Tumor

2.7

71

Lung Normal

2.955

Lung Tumor

3.250

Colon Normal

N/A32

Colon Tumor

N/A39

Stomach Tumor

2.430

Stomach Normal

2.68*The “DV200” is the percentage of RNA fragments > 200 nucleotides. Although RIN values
for these samples lie within a relatively narrow range (2.3–3.2), the size distribution of theRNA varies greatly among the samples.

Figure 2: RNA Quality from FFPE Samples— RNA isolated from FFPEsamples was examined using an Agilent Bioanalyzer. RNA Integrity Numbers(RINs)5 were calculated from the Bioanalyzer traces.

APre-Capture Library Yield
1000

Evaluating RNA Quality

800

The DV200 value can be easily calculated from Fragment Analyzer or
Bioanalyzer traces (Figure 5). Customized Illumina FFPE RNA DV200
Fragment Analyzer methods for standard sensitivity and high sensitivity
RNA sample kits can be downloaded from the Advanced Analyticalwebsite at www.aati-us.com/product/fragment-analyzer.

600
400
200
0

RIN:BNBTLNLTCTCNST

SN

2.3

2.7

2.9

3.2

NA

NA

2.6

2.4

B
1000

800

Yield (ng)Because mean RNA fragment size is a more reliable determinantof RNA quality for the TruSeq RNA Access Library Preparation Kit(Figure 3), Illumina developed the DV200 metric—the percentage ofRNA fragments > 200 nucleotides. Using DV200 to assess FFPE
RNA quality enables accurate fine-tuning of the minimal RNA input
required for successful library preparation. By adjusting RNA input
amounts, high-quality libraries can be prepared from poor-quality FFPE
samples (Table 2). The recommended quantities of starting material
correspond to a range of DV200 values; note that using RNA samples
with DV200 < 30% is not recommended (table 2). Using the DV200
metric to determine the RNA input requirement also ensures robust,
reproducible results. Excellent correlation (R2=0.99) was observed
between the normalized gene expression counts from 3 different FFPE
tumor samples of varied RNA quality (Figure 4).

Yield (ng)Many researchers use the Agilent RNA Integrity Number (RIN)5
to determine RNA quality for gene expression analysis (Figure 2).
However, we have found that RIN values from degraded FFPE
samples are not a sensitive measure of RNA quality. They are also
not a reliable predictor of successful library preparation (Table 1and Figure 3).

60040020000255075100DV200Figure 3: RIN versus DV200 and Library Yield—(A) Libraries were prepared from20 ng total RNA from 8 FFPE samples. RIN values range from 2.3 – 3.2 and do
not show a correlation to precapture library yield. (B) High correlation (R2 = 0.91)
between precapture library yield and DV200.

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

Technical Note: RNA Sequencing

Figure 4: High concordance of gene expression between replicates across FFPE samples of varied quality—TruSight RNA Pan-Cancer libraries were preparedfrom High, Medium, and Low Quality FFPE from 20 ng, 50 ng, and 100 ng of total RNA, respectively. Normalized gene expression counts, in fragments per kilobase per
million mapped reads (FPKM), were calculated by BaseSpace® TopHat Alignment App.4

A

Table 2: Recommended RNA Input Based on DV200

1Recommended Input Quantity
ParameterQuality*†‡

DV200

TruSeq RNAAccess

TruSight RNA
Pan-Cancer or
TruSight RNAFusion

High> 70%

20 ng

20 ng

Medium50–70%

20-40 ng

20-50 ng

Low

30–50%

40–100 ng

50–100 ng

Too Degraded< 30%23Not Recommended* to ensure successful library prep, we recommend using an RNA isolation method thatincludes a reverse-crosslinking step and DNase1 treatment (we used QIAGEN RNeasy
FFPE Kit or QIAGEN AllPrep DNA/RNA FFPE Kit).
† FFPE RNA concentration was determined by NanoDrop.
‡ To ensure best performance on samples close to edge of a quality classification, err
towards the higher end of the input recommendation.

4B

Summary
Next-generation sequencing approaches applied to FFPE preserved
tissue samples along with their associated clinical data offer aninvaluable resource for translational research. The RNA enrichment
library prep kits allow researchers to utilize FFPE and other challenging
samples in their NGS studies. For these studies, the DV200 of RNAfragments, as determined by a Fragment Analyzer or Bioanalyzer
trace, is a reliable predictor for successful RNA sequencing results
using the TruSeq RNA Access Library Preparation Kit, or the TruSight
RNA Pan-Cancer Panel.

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

1

Figure 5: Calculate DV200—(A) The percentage of RNA fragments > 200 nt(DV200) can be calculated from a Bioanalyzer trace by performing a Smear Analysis
as follows: 1) Under the Local tab, change Normal to Advanced. 2) Check box
for Smear Analysis. 3) Click Table, add a region, and enter 200–10,000 bp in thepopup window. 4) Select the Region Table tab in the trace window to display theresults. (B) The Fragment Analyzer system has a streamlined solution for DV200
analysis. Prosize™ software automatically configures the > 200 nt smear analysis
parameters, and the DV200 result is displayed as the% Total value within the data table.

Technical Note: RNA SequencingOrdering Information

References
1.

Product

Catalog No.

TruSight RNA Pan-Cancer Panel
Set A (48 samples, 12 indexes)

RS-303-1002

TruSight RNA Pan-Cancer Panel
Set B (48 samples, 12 indexes)

RS-303-1003

TruSight RNA Fusion Panel
Set A (48 samples, 12 indexes)

RS-304-1002

TruSight RNA Fusion Panel
Set B (48 samples, 12 indexes)

RS-304-1003

TruSeq RNA Access Library Prep Kit
Set A (48 samples, 12 indexes)

RS-301-2001

TruSeq RNA Access Library Prep Kit
Set B (48 samples, 12 indexes)

RS-301-2002

2.

3.

4.

5.

von Ahlfen S, Missel A, Bendrat K, and Schlimpberger M. Determinants of
RNA quality from FFPE samples. PLoS ONE 2007;2(12): e1261.
Penland SK, Keku TO, Torrice C, He X, Krishnamurthy J, Hoadley KA, etal. RNA expression analysis of formalin-fixed paraffin-embedded tumors.
Lab Invest 2007;794: 383–391.
Norton N, Sun Z, Asmann YW, Serie DJ, Necela BM, et al. Gene expression,single nucleotide variant and fusion transcript discovery in archival material
from breast tumors. PLOS One 2013;8(11): e81925.
Illumina. BaseSpace Core Applications: TopHat Alignment App. support.
illumina.com/sequencing/sequencing_software/basespace/documentation.
ilmn. Accessed May 25, 2016.
Agilent Technologies. (2004) RNA Integrity Number (RIN) - Standardizationof RNA Quality Control Publication PN 5989-1165EN. www.chem.agilent.
com. Accessed May 25, 2016.

Learn MoreFor more FFPE RNA solutions, visit
www.illumina.com/applications/sequencing/rna/low-quality-ffpe-rnaseq.ilmn
To learn more about Illumina RNA enrichment assays, visit
TruSight RNA Pan-Cancer: www.illumina.com/RNAPanCancer
TruSight RNA Fusion: www.illumina.com/RNAFusion
TruSeq RNA Access: www.illumina.com/products/truseq-rna-accesskit.ilmn

Illumina • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
For Research Use Only. Not for use in diagnostic procedures.
© 2016 Illumina, Inc. All rights reserved. Illumina, BaseSpace, TruSeq, TruSight, and the pumpkin orange color are trademarks of Illumina,
Inc. and/or its affiliate(s) in the U.S. and/or other countries. Pub. No. 470-2014-001 Current as of 26 October 2016