MicroRNAs (miRNA) are a class of small non-coding RNAs (approximately 21 nt long) that bind complementary sequences in target mRNAs to specifically regulate gene expression. Aberrant regulation of miRNAs and their targets has been associated with several diseases including cancer. The relationship between miRNA and mRNA has been found to be important in cancer development and progression. Simultaneous expression studies of miRNA and mRNA and detection of mutations in mRNA transcripts can be valuable in understanding molecular mechanisms that have an underlying role in various diseases. We demonstrate the technical verification of a novel method to reverse-transcribe and pre-amplify miRNA and mRNA from sample-limiting serum research samples using the TaqMan® Advanced miRNA cDNA Synthesis Kit. Based on results from previous studies, a signature of 49 mRNA and 37 miRNA targets has been identified that may help distinguish between benign and malignant pancreatic tissues. In this study, these targets and an additional set of transcript mutations were analyzed in serum from normal and test samples. TaqMan assays for miRNA and mRNA targets and custom TaqMan Mutation Detection Assays (TMDAs) were placed on TaqMan Array Cards to facilitate investigation of several samples in a single experiment. Results demonstrate that transcript mutations can be detected and miRNA and mRNA targets can be reliably quantified from a single reverse transcription reaction. For research use only. Not for use in diagnostic purposes.

1. Thermo Fisher Scientific • 180 Oyster Point Blvd • South San Francisco, CA 94082 • thermofisher.com
mRNA/miRNA Reverse
Transcription
Component
Per
Sample
(µl)
5X RT Buffer 6.0
dNTP Mix, 100mM 1.2
Random Hexamers 1.0
20X Universal RT
Primer
1.5
10X RT Enzyme Mix 3.0
Nuclease-free water 2.3
Total 15.0
Reverse Transcription
Step Temp Time
Reverse
Transcription 42oC 15 min
Stop
reaction 85oC 5 min
Hold 4oC ∞
RESULTS
Mutations frequently found in pancreatic cancer were selected for analysis using custom TMDAs
targeting cDNA. Several candidate assays for each mutant and non-mutant cDNA were designed by the
custom assay service at Thermo Fisher Scientific and were tested with synthetic templates to determine
which performed best in terms of specificity and dynamic range. Control research serum samples were
processed using the described protocol, and the resulting preamplified cDNA was analyzed to determine
a DCRT cutoff for percent detection of mutant transcripts. The assays were then used to analyze research
serum samples from individuals with pancreatic cancer on TaqMan Array Cards to determine the percent
of mutant transcript. Amplification data from two control vs. two cancer samples are shown above. The
greater relative abundance of mutant vs. non-mutant transcript is visible in the plots as a smaller gap
between reference and mutant assay amplification curves in the data from cancer samples vs. controls.
Figure 6. Custom TaqMan Mutation Detection Assay Amplification Plots
Figure 5. Unsupervised clustering of mRNA and miRNA expression
Kathleen C. Hayashibara1, Harita Veereshlingam1, Malte Buchholz2
1Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA 94080. 2Philipps-Universitaet Marburg, 35043 Marburg, Germany.
ABSTRACT
MicroRNAs (miRNA) are a class of small non-coding RNAs (approximately 21 nt
long) that bind complementary sequences in target mRNAs to specifically regulate
gene expression. Aberrant regulation of miRNAs and their targets has been
associated with several diseases including cancer. The relationship between miRNA
and mRNA has been found to be important in cancer development and progression.
Simultaneous expression studies of miRNA and mRNA and detection of mutations in
mRNA transcripts can be valuable in understanding molecular mechanisms that
have an underlying role in various diseases. We demonstrate the technical
verification of a novel method to reverse-transcribe and pre-amplify miRNA and
mRNA from sample-limiting serum research samples using the TaqMan® Advanced
miRNA cDNA Synthesis Kit. Based on results from previous studies, a signature of
49 mRNA and 37 miRNA targets has been identified that may help distinguish
between benign and malignant pancreatic tissues. In this study, these targets and an
additional set of transcript mutations were analyzed in serum from normal and test
samples. TaqMan assays for miRNA and mRNA targets and custom TaqMan
Mutation Detection Assays (TMDAs) were placed on TaqMan Array Cards to
facilitate investigation of several samples in a single experiment. Results
demonstrate that transcript mutations can be detected and miRNA and mRNA
targets can be reliably quantified from a single reverse transcription reaction.
For research use only. Not for use in diagnostic purposes.
INTRODUCTION
Purified total RNA from serum, containing a mixture of miRNAs and mRNAs, is
purified using the mirVana PARIS kit, then converted to cDNA using two different
methods: 1) The TaqMan Advanced miRNA cDNA Synthesis Kit to analyze miRNA
and mRNA, 2) The High Capacity cDNA Synthesis Kit to detect mutant transcripts.
In the TaqMan Advanced miRNA cDNA Synthesis Kit protocol, PolyA polymerase is
used to add a 3’ adenosine tail to the miRNAs and mRNAs, polyA-tailed miRNAs but
not mRNAs then undergo a 5’ adaptor ligation. This 5’ adaptor acts as the forward
primer binding site in subsequent miR-Amp reactions. Universal RT primer binds to
the 3’poly (A) tail of miRNA and mRNAs and converts it to cDNA. Reverse
transcription of mRNAs is further facilitated by random hexamers. The resulting
cDNA undergoes a universal miR-Amp reaction and gene specific pre-amplification
reaction for 14 cycles in a single tube. A separate reverse transcription reaction
using the High Capacity cDNA Synthesis Kit is used to create large amounts of
cDNA to detect mutant transcripts that are present at low frequency relative to non-
mutant transcripts. This material is then concentrated and preamplified separately
for 22 cycles to obtain enough mutation-containing cDNA for detection. Resulting
products from both preamplification reactions are pooled and detected by qPCR
using gene-specific TaqMan Gene Expression Assays or miRNA-specific TaqMan
Advanced miRNA Assays or cDNA-specific TaqMan Mutation Detection Assays
(TMDA) based on Competitive Allele-Specific TaqMan PCR (castPCR) technology.
MATERIALS
Samples: Research serum samples from apparently normal individuals or individuals
with stage 4 pancreatic cancer were obtained from BioIVT. Custom TaqMan Array
Cards: Custom TaqMan® Array Card (format 96a, Cat. No. 4342259). RNA
extraction: mirVana™ PARIS™ RNA and Native Protein Purification Kit (Cat. No. AM
1556). cDNA synthesis: For mRNA and miRNA analysis, total RNA was reverse-
transcribed using the TaqMan advanced miRNA cDNA synthesis Kit (Cat. No.
A28007) with the addition of random hexamers to enable detection of mRNA. For
TMDA experiments total RNA was reverse-transcribed using the High-Capacity
cDNA Reverse Transcription Kit (Cat. No. 4368814). TaqMan™ PreAmp Master Mix
(Cat. No. 4391128) was used for preamplification of TMDA targets. TaqPath™
ProAmp™ Master Mix (Cat. No. A30871) was used for qPCR.
CONCLUSIONS
We have developed a thermal protocol for analyzing mRNA, miRNA and for mutation
detection by qPCR on the same TaqMan Array Card using TaqMan Gene Expression
Assays, TaqMan Advanced miRNA Assays, and custom TaqMan Mutation Detection
Assays.. The protocol also allows a single qPCR reaction mixture to be loaded onto
the card for a simplified workflow. The information obtained from these assays may
help to distinguish between benign and malignant pancreatic tissues.
REFERENCES
1. Gress TM, Lausser L, Schirra LR, Ortmüller L, Diels R, Kong B, Michalski CW,
Hackert T, Strobel O, Giese NA, Schenk M, Lawlor RT, Scarpa A, Kestler HA,
Buchholz M. Combined microRNA and mRNA microfluidic TaqMan array cards for the
diagnosis of malignancy of multiple types of pancreatico-biliary tumors in fine-needle
aspiration material. Oncotarget. 2017; 8(64):108223-108237.
2. Buchholz M, Kestler HA, Bauer A, Bock W, Rau B, Leder G, Kratzer W, Bommer M,
Scarpa A, Schilling MK, Adler G, Hoheisel JD and Gress TM. Specialized DNA arrays
for the differentiation of pancreatic tumors. Clin Cancer Res. 2005; 11(22):8048-8054.
3. Gress TM, Kestler HA, Lausser L, Fiedler L, Sipos B, Michalski CW, Werner J,
Giese N, Scarpa A and Buchholz M. Differentiation of multiple types of pancreatico-
biliary tumors by molecular analysis of clinical specimens. JMolMed (Berl). 2012;
90(4):457-464.
4. van der Poel HG, Oosterhof GON, Schaafsma HE, Debruyne FMJ, Schalken JA.
Intratumoral nuclear morphologic heterogeneity in prostate cancer - Urology 1997
49(4):652-657.
5. Mestdagh P, Van Vlierberghe P, De Weer A, et al. 2009. A novel and universal
method for microRNA RT-qPCR data normalization. Genome Biology 10, R64.
ACKNOWLEDGEMENTS
We thank Kelly Li, Stephen Jackson, Fangqi Hu, Linda Wong and Shoulian Dong for
their technical input and Xiaoqing You for custom assay designs.
TRADEMARKS/LICENSING
© 2017 Thermo Fisher Scientific Inc. All rights reserved All trademarks are the
property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under
permission and license.
For Research Use Only. Not for use in diagnostic procedures.
TaqMan® Advanced miRNA cDNA synthesis kit to simultaneously study expression of
miRNA and mRNA and detect mutations in serum samples
METHODS Figure 2. RT of mRNA and miRNA into
cDNA and cDNA amplification
The TaqMan Advanced cDNA Synthesis Kit is used
to reverse-transcribe both mRNA and miRNA into
cDNA and amplify cDNA using target-specific
primers for mRNA and universal primers for miRNA.
Table 1. TaqMan Advanced cDNA
Synthesis Kit Protocol with Modifications
Figure 1. Sample Workflow
Total RNA is isolated from 1 ml of serum using
the mirVana™ PARIS™ RNA and Native Protein
Purification Kit using the standard protocol for
liquid samples. The total RNA is processed as
shown above in two different paths, the top path
for mRNA and miRNA analysis and the bottom
path for mutation detection. The samples from
both paths are then pooled for analysis on a
TaqMan® Array Card (TAC).
Figure 3. RT of mRNA and amplification
of cDNA for TMDA experiments
The High Capacity cDNA Reverse Transcription Kit
is used to reverse-transcribe mRNA into cDNA.
The cDNA is preamplified for 22 cycles using pooled
target-specific primers spanning mutation-containing
regions of the targets of interest.
miR-Amp and Pre-Amp
Component
Per
Sample (µl)
2X miR-Amp Master
Mix
25.0
20X miR-Amp Primer
Mix
2.5
0.2X Pooled mRNA
assays
12.5
Nuclease-free water 5.0
Total 45.0
miR-Amp and Pre-Amp Cycling
Step Temp Time Cycles
Enzyme
Activiation 95oC 10min
Denature 95oC 15sec 14
Anneal/
Extend 60oC 4min 14
Stop 99oC 10min
Hold 4oC ∞
Table 2. TaqMan Mutation Detection Assay target preparation
High-Capacity cDNA Reverse
Transcription Kit for TMDA Analysis
Component
Per
Sample
(µl)
10x RT Buffer 20
25x dNTP Mix (100 mM) 8
1x RT Random Primers 20
MultiScribe Reverse
Transcriptase
10
RNA 142
Total 200
Reverse Transcription
Step Temp Time
1 25oC 10 min
2 37oC 2 hrs
3 85oC 5 min
4 4oC ∞
TMDA cDNA
Preciptation/Concentration
Component
Per
Sample
(µl)
Reverse Transcription
Reaction
200
3.0 M Sodium Acetate,
pH 6.0
20
100% Ethanol 600
Total 820
Precipitate at -20oC for 1 hour.
Wash pellet with 1 ml 70%
ethanol/30% nuclease-free water
Air-dry pellet and resuspend in 20 µl
nuclease-free water
TMDA cDNA Preamplification
Component
Per
Sample
(µl)
2X TaqMan Preamp
Master Mix
40
Concentrated cDNA 20
9 nM target loci
primer pool
20
Total 80
Preamplification Cycling
Step Temp Time Cycles
Enzyme
Activiation 95oC 10min
Denature 95oC 15sec 22
Anneal/
Extend 60oC 4min 22
Stop 99oC 10min
Hold 4oC ∞
cDNA TaqMan Array Card
qPCR
Component
Per
Sample
(µl)
TaqPath ProAmp
Master Mix
50
1:5 diluted TMDA
preamp reaction
25
1:10 diluted miR-
AMP with mRNA
(Table 1)
25
Total 100
(See Figure 4 for thermal
protocol)
Based on previous studies, a signature of mRNA and miRNA targets was identified that may help to
distinguish between benign and malignant pancreatic tissues [1-5]. Preamplified cDNA derived from
miRNA and mRNA targets were mixed in a qPCR reaction with pooled amplified potential mutation-
containing loci for TMDA analysis. The Thermo Fisher Cloud RQ app was used to generate a heat map to
cluster targets and samples using Pearson’s Correlation, Complete Linkage, and Target-Centric settings.
Pancreatic cancer research samples cluster (prefix PC) cluster outside of control samples (prefix CTRL)
with the exception of sample PC_3. All PC samples were from individuals under treatment with various
chemotherapy agents, which may affect gene expression. Control samples are from apparently normal
individuals who may or may not be cancer-free.
Table 3. Percent mutation analysis of pancreatic cancer research samples
Mutation/Sample PC_1 PC_2 PC_3 PC_4 CTRL_1 CTRL_2 CTRL_3
KRAS G12D 0 0 (0.082) 0 0 0 0
KRAS G12C 0 0.28 0 0 0 0 0
KRAS G12V 0 0 (0.082) 0.13 0 0 0
TP53 R175H 0 0 0 0 0 0 0
Pancreatic cancer research samples were analyzed for four commonly-occurring mutations by
processing data from custom TaqMan Mutation Detection Assays targeting these sites in KRAS and
TP53 cDNA using Mutation Detector™ Software v2.0. The serum samples were isolated from individuals
with stage 4 pancreatic cancer. Mutant transcripts were detected in two of the research serum samples
with the percentages indicated above. Sample PC_2 had the highest tumor load. The analyzed
mutations were not detected in samples PC_1 or PC_3, where mutant transcripts for these targets may
be below the detection limit of 0.10%.
Control
Sample
Pancreatic Cancer
Sample
KRAS Ref
Assay
KRAS Ref
Assay
KRAS
G12V
Assay
KRAS
G12V
Assay
Control
Sample
Pancreatic Cancer
Sample
KRAS Ref
Assay
KRAS Ref
Assay
KRAS
G12D
Assay
KRAS
G12D
Assay
PC_3 CTRL_1 CTRL_2 CTRL_3 PC_2 PC_1 PC-4
Figure 4. TaqMan Array Card thermal protocol