1. Improved RNA Quality from Formalin-Fixed Paraffin Embedded Tissue
Using ArrayGrade™ FFPE RNA Isolation Kit
Savita Prabhakar, Hewen Zhang, Yexun (Bill) Wang, and Xiao Zeng. SuperArray Service Core. 7320 Executive Way, Suite 101; Frederick MD 21704.
Introduction
Formalin-Fixed Paraffin Embedded (FFPE) samples are the most readily available material from
human disease studies for which pathology and clinical outcomes are well documented. Analyzing
gene expression patterns or “profiles” in these archived tissues should facilitate retrospective studies
correlating gene expression patterns with cancer progression. However, RNA extracted from FFPE
blocks is often too heavily cross-linked to effectively serve as a substrate for subsequent labeling
reactions. This study describes a new kit for isolating RNA from FFPE blocks and slides that
improves gene expression profiling sensitivity and the number of positive calls obtained in both highdensity genome-wide and low-density pathway-focused microarray formats. The key to enhanced
sensitivity is a more effective reversal of the chemical cross-links from RNA caused by formalin
fixation. The ArrayGrade™ FFPE RNA Isolation Kit more readily isolates RNA from fixed tissues
and provides a better template for microarray labeling protocols or real-time PCR than other kits.
Data presented here demonstrates that this method isolates RNA from FFPE samples with greater
yield, purity, and real-time PCR sensitivity. Most importantly, the RNA isolated by this method also
provides percent positive calls and real-time PCR threshold cycle values that are more comparable to
fresh tissue samples using SuperArray’s RT2Profiler™ PCR Arrays and Oligo GEArrays® as well as
the Agilent DNA microarrays. The ArrayGrade™ FFPE RNA isolation method now brings both
microarray-based and real-time PCR profiling to researchers with access to archived tissue samples.
Performance of ArrayGrade™ FFPE RNA on RT2 Profiler™ PCR Arrays
ArrayGrade™
Profiler™
Performance of ArrayGradeTM FFPE RNA on Microarrays
Figure 2: ArrayGrade™ FFPE RNA Yields More Positive Calls, Improves Sensitivity, and Provides
ArrayGrade™
Expression Profiles Comparable to Fresh Tissue on Genome–Wide Agilent Microarrays
Genome–
Figure 4: ArrayGrade™ FFPE RNA Yields Greater Real-Time PCR Sensitivity on PCR Arrays
ArrayGrade™
Realusing the RT²Profiler™ Mouse Tumor Metastasis PCR Array as an Example
RT² Profiler™
Percentage of Genes
A
Kit ‘’I’’
‘’I
RNA samples isolated from sections of a mouse kidney FFPE block, with either ArrayGrade™ or two other
commercial methods, and RNA from fresh tissue were characterized on the Mouse Oligo Microarray Kit (V2)
from Agilent according to the manufacturer’s instructions. Representative images from the same sections of
each microarray are displayed with a few spots circled to emphasize the relative intensity level of these genes
observed in each RNA sample.
32.7
2.0 √
11.8
2.0 √
4.2
2.0 √
1.1
Kit “I”
4.6
1.8 √
1.2
Liver
5 20- μ sections @
0.4 cm x 0.8 cm
each
Yield (μg)
A260:A280
A260:A230
ArrayGrade
26.2
2.1 √
2.2 √
Kit “A”
11.5
2.0 √
2.0 √
6.1
1.8
1.0
Yield (μg)
A260:A280
A260:A230
7.0
2.0 √
1.1
Kit “Q”
Kit “I”
Heart
3 20- μ sections @
0.4 cm x 0.5 cm
each
ArrayGrade
ND
Kit “A”
ND
Kit “Q”
1.9
1.5
1.0
1.6
0.7
25-30
30-35
26684
N/A
8766
4868
y = 0.724x – 0.220 R2 = 0.385
7909
3068
y = 0.583x + 0.106 R2 = 0.331
Kit “I”
2356
1127
y = 0.376x – 0.314 R2 = 0.175
40%
30%
20%
10%
0%
25-30
30-35
40%
30%
20%
10%
0%
<25
25-30
30-35
Figure 5: Identifying Differentially Expressed Genes Between FFPE Blocks of Different Tissues using the
FFPE
RT²Profiler™ Human Insulin Signaling Pathway PCR Array
RT² Profiler™
A
Gene
The percent positive calls from the arrays in Panel A were calculated and expressed as either “Significantly
Above Background”, meaning the number of mean signal intensities above background based on a two-sided ttest, or “Well Above Background”, meaning the number of background-corrected signal intensities greater than
2.6 standard deviations about the average background. The fresh tissue signal intensities were plotted against
the intensities from each FFPE RNA isolation method in separate scatter plots (data not shown). The curve fits
to a straight line were determined and also listed in the table highlighting how close the slopes and the
correlation coefficients are to the ideal of 1.0.
Fold Change
0.00008
IRS4
173.69
0.00028
LEP
109.93
0.00243
TG
101.62
0.00100
GCK
72.46
Figure 3: ArrayGrade™ FFPE RNA Provides More Positive Calls and Better Sensitivity on
ArrayGrade™
Oligo GEArray® than Other Commercial Methods.
GEArray®
0.00013
RETN
57.25
0.00005
DOK2
53.80
0.00016
UCP1
29.13
0.00031
DOK3
21.02
0.00042
17.64
0.00044
CBL
15.30
0.00067
RRAS
13.87
0.00004
DOK1
12.74
0.00131
HRAS
12.74
0.00027
SREBF1
A
11.33
0.00007
NOS2A
Figure 1: Size of RNA Isolated from ArrayGrade™ is More Compatible with Microarray Analysis
ArrayGrade™
than Other Commercial Kits
Kit ‘’Q’’
‘’Q
Kit ‘’A’’
‘’A
Kit ‘’I’’
‘’I
B
Kit “I”
Percent Positive Calls
ArrayGrade
Kit “A”
RNA Isolation Method
Kidney
Heart
Liver
Fresh
52.9
42.9
ArrayGrade
23.5
30.2
Kit “Q”
20.2
18.5
ND
Kit “A”
16.0
ND
22.7
Kit “I”
The size distribution of RNA isolated from sections of a mouse kidney FFPE block using the
kidney
ArrayGrade™ Total RNA Isolation Kit as well as Kits “A”, “I”, and “Q” was determined with RNA
ArrayGrade™
NanoChip 6000 on an Agilent BioAnalyzer. RNA isolated from the ArrayGrade™ kit is neither heavily
ArrayGrade™
cross-linked (unlike RNA from Kit “A”) nor smaller in molecular weight (unlike RNA from Kits “Q” and
cross“I”). The ArrayGrade™ RNA size range provides a middle ground between no heat treatment (Kit “A”)
ArrayGrade™
treatment (Kit
and harsh heating procedures (Kits “Q” and “I”).
(Kits
0.00077
3.52
Sple e n
TG
IRS4
100.00
1.E-05
INS
RRAS
GCK
1.E-04
LEP
RETN
HRAS
FBP1
10.00
1.00
INS
GCK
IRS4
TG
1.E-03
1.E-02
1.E-01
FBP1
0.10
12 11
10
9
1.E+00
Inte stine
8
7
Column
6
5
4
3
2
1
A
B
C
D
E
F
G
H
-5
-3
-1
1
3
5
7
9
Log2(Fold Difference)
Row
0.00092
-8.20
1000.00
0.00070
RNA isolated from human spleen and intestine FFPE blocks using the ArrayGrade™ RNA Isolation Kit was
characterized on the RT2Profiler™ Human Insulin Signaling Pathway PCR Array (APHS-030). Out of the 84
insulin pathway focused genes, 35 genes demonstrate at least a three-fold difference in gene expression
between spleen and intestine tissues. Three RNA isolations were done from each block, and all the three
technical RNA isolation replicates were run on individual PCR Arrays. Panel A lists the 18 of the
differentially expressed genes with a p value less than 0.001. Six of these genes (colored red) are associated
with both insulin signaling and lipid metabolism. Panel B plots the fold changes of each gene as a z-axis
displacement from the xy-plane representing the 96-well layout of the PCR Array. The volcano plot (panel C)
depicts the log2 transformation of the fold-changes versus the p-values from the student t-test of the triplicates.
Conclusions
37.8
24.4
9.75
FBP1
RNA was isolated from replicate sets of sections cut from FFPE blocks of mouse kidney, liver, or heart
blocks
organs using the ArrayGrade™ method or either two or three other commercial kits (“A”, “Q”, or “I”).
ArrayGrade™
(“
The yields and purities, expressed as A260:A280 and A260:A230 ratios, of the RNA samples are listed.
ratios,
Absorption ratios with a check mark (√) pass while those without fail the quality control necessary for
(√
for
microarray analysis. ND (Not Determined).
C
p Value
384.97
B
TTest
Spleen vs Intestine
INS
CEBPB
ArrayGrade
Absent Calls
Threshold Cycle Value Range
Symbol
Fresh
Absent Calls
Threshold Cycle Value Range
50%
PIK3R2
Kit “Q”
Absent Calls
50%
<25
Percentage of Genes
31791
0.8
Kit “I”
RNA samples isolated from sections of a mouse
kidney FFPE block using either ArrayGrade™ or
another commercial kit “A” or RNA from fresh
mouse kidney were converted to cDNA template
using the ReactionReady™ First Strand cDNA
ArrayGrade™ Synthesis Kit (C-01). PCR was performed on the
Bio-Rad iCycler® using the RT2 Real-Time™
SYBR / Fluorescein PCR Master Mix (PA-011) and
the RT²Profiler™ Mouse Tumor Metastasis PCR
Array (APMM-028). The threshold cycle (Ct) value
distribution for all 84 genes on the array in all three
Kit “A”
RNA samples are displayed.
0%
Correlation with Fresh Tissue
2.0 √
Kit “Q”
Well Above
Background
Kit “Q”
1.7 √
Kit “A”
Significantly
Above
Background
Fresh
A260:A230
ArrayGrade
Fresh
10%
p Value
Kit ‘’Q’’
‘’Q
ArrayGrade
5 20-μ sections @
1.0 cm x 0.5 cm
each
20%
Fold Difference ( Spleen/ Intestine)
ArrayGrade
Percentage of Genes
Fresh
Table 1: Improved RNA Isolation: Greater Quantity and Quality than Other Commercial Kits
than
A260:A280
30%
Threshold Cycle Value Range
RNA
Isolation
Method
Yield (μg)
40%
<25
B
Kidney
50%
2.5
26.0
6.7
Equal amounts of RNA (3 μg) isolated either from fresh mouse kidney or FFPE mouse kidney sections using
the ArrayGrade™ method and three other commercial kits were used to generate cRNA with the TrueLabelingAMP™ 2.0 Kit (GA-030) in an overnight reaction. Labeled cRNA (3.0 μg) was hybridized overnight to
identical copies of the Mouse Tumor Metastasis Oligo GEArray® in the HybPlate Format (EMM-028). Panel
A: The raw microarray images are displayed demonstrating more visible spots from the RNA isolated by the
ArrayGrade™ method than the other commercial methods. A few spots are circled to emphasize the relative
intensity level of the genes observed in each RNA sample. Panel B: The percent present call for each sample is
displayed as determined by the GEArray Expression Analysis Suite.
The ArrayGrade™ FFPE RNA isolation method yields RNA of sufficient quality and quantity from
FFPE blocks and slides for PCR and microarray analysis. The method also …
method
Uses a unique and proprietary method that effectively removes chemical cross-links and improves
chemical crossRNA sensitivity.
Increases present positive calls and sensitivity on pathway-focused DNA microarrays and genome–
pathwaygenome–
wide microarrays.
Lowers real-time PCR threshold cycle values and can be reliably used with PCR Arrays, a
realtechnology that offers a simple and reliable method to identify and monitor gene expression
changes within a defined group of genes.
Enables researchers with access to archived tissue samples to perform gene expression profiling
reliably.