Technical Guide to

QIAGEN PCR Arrays

RT2 Profiler PCR Arrays

RT2 lncRNA PCR Arrays
miScript PCR Arrays

Sample to Insight
Total RNA discovery with RT2 and
miScript PCR Arrays
Explore the RNA universe
Whatever your destination within the RNA universe, QIAGEN will help you get there. The
miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and
miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA
expression and for validation of microarray and RNA sequencing experiments. Together with the
powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products
give you a smooth path from your sample to high-quality results.

From one sample – the whole range of RNA results

With our workflow (Figure 1), you can take any single sample and discover the secrets of the
messenger RNA (mRNA), long non-coding RNA (lncRNA) and microRNA (miRNA). Whether
you’re studying serum or plasma samples, FFPE tissues, cultured cell lines or exosomes, the kits are
optimized to provide excellent results – even with limited amounts of sample.

miScript miScript
miScript II
PreAmp miRNA
RT Kit
miRNA PCR Kit PCR Array

Sample type Isolation Analytics

Serum or Optional QIAGEN

GeneGlobe
plasma miRNeasy preamplification Ingenuity
RT reaction PCR array Data
FFPE tissues Kit for low sample Pathway
Analysis
inputs Center Analysis
Cell lines
Exosomes

mRNA RT2 PreAmp

lncRNA RT2 First RT2 Profiler/
Strand cDNA lncRNA
cDNA Synthesis PCR Array
Kit

Figure 1. From any sample to pathway insights – the total RNA workflow from QIAGEN.

2  Technical Guide to QIAGEN PCR Arrays 06/2016

Laboratory-validated real-time PCR-based RNA quantification
and verification
Real-time PCR provides flexibility and speed for time-critical assays. It is universally recognized as
the best means of quantifying and verifying the contents of both annotated and novel mRNA,
miRNA and lncRNA. Its success relies on having the right technology to give:

• Sensitivity – assays designed to efficiently detect and quantify even the rarest target

• Specificity – on-target amplification from each single assay to the full array

• Dynamic range – reliable with large or small input volumes, robust or rare targets, one assay
or thousands

QIAGEN is a world-renowned supplier of sensitive, specific and dynamic, laboratory-verified PCR-

based solutions that address all the requirements of RNA profiling work.

miRNeasy Mini Procedure

Start the easy way – with miRNeasy Cells/tissue

and exoRNeasy
Lyse and
homogenize
Get total RNA from every sample
Our miRNeasy Kits isolate pure, high-quality total RNA, including mRNA,
miRNA and lncRNA, from cells, easy- and difficult-to-lyse tissues, FFPE Add chloroform
and shake
samples and all biofluids including serum, plasma, CSF, urine and cell
Separate phases
culture media.

These kits enable efficient enrichment of RNA down to approximately

18 nt in size, even when low amounts of starting material are used. This
Add ethanol to
versatility and quality is achieved thanks to phenol/guanidine-based lysis aqueous phase

of samples followed by silica membrane-based purification (Figure 2). Bind total RNA
including small RNA

The power of miRNeasy lysis

The QIAzol® Lysis Reagent included in the kit is a monophasic solution of
phenol and guanidine thiocyanate. It is designed to facilitate tissue lysis, Wash
inhibit RNases and remove most of the cellular DNA and proteins from
the lysate by organic extraction. Elute

Total RNA including small RNAs

Figure 2. The miRNeasy workflow.

Technical Guide to QIAGEN PCR Arrays 06/2016  3

High-purity RNA from FFPE tissues
The crosslinking and fragmentation that occurs during the FFPE process can make purification
of nucleic acids challenging. The miRNeasy FFPE Kit provides special lysis and incubation
conditions to reverse formalin crosslinking of RNA, giving efficient purification without further
degradation. To remove even trace amounts of DNA, which can impair RT-PCR, the RNA is treated
with both DNase and DNase Booster Buffer.

Circulating miRNA purification from serum or plasma

With their potential to serve as biomarkers for the detection of cancers and other diseases,
circulating miRNA are desirable assay targets. The miRNeasy Serum/Plasma Kit enables total RNA
purification from even very small sample volumes. The miRNeasy Serum/Plasma Spike-In Control
supports normalization when working with such samples.

What about exosomes?

Exosomes also have considerable potential in the study of cancers and other diseases. Using
similar principles of purification of total RNA, we developed the exoRNeasy Serum/Plasma Maxi
Kit, which provides microvesicle isolation in just 20 minutes (Figure 3). A subsequent 35-minute
isolation procedure yields total RNA from these extracellular vesicles. Just one hour from sample
to microvesicle RNA means you can spend less time getting the RNA and more time deciphering
what it means.

Add chloroform
to QIAzol eluate
Mix sample with
Buffer XBP and
bind to column
Spin blood Recover aqueous
phase and add
ethanol
Transfer
plasma/serum

Bind total RNA

Wash bound including miRNAs
exosomes with
Buffer XWP

Filter

Wash 3x

Elute

Filtered Lyse vesicles and

plasma/serum elute with QIAzol Total RNA
Figure 3. The exoRNeasy
including miRNAs
Serum/Plasma Maxi Kit
workflow.

4  Technical Guide to QIAGEN PCR Arrays 06/2016

Profiling coding and noncoding RNA from research samples

RNAs by pathway & disease Convert total RNA to cDNA

Unlock the secrets of the transcriptome

RT2 Profiler and RT2 lncRNA PCR Arrays are highly reliable and sensi-
tive technologies for analyzing focused panels of genes to discover their
roles in signal transduction, biological processes or disease pathways. Add cDNA to RT2 SYBR® Green qPCR Mastermix
Aliquot mixture across RT2 Profile PCR Array
They use a straightforward workflow based on RT-PCR (Figure 4) and can
Run using qPCR instrument
identify basal and up- or downregulated expression of genes (Figure 5).

Each PCR Array contains a list of pathway-focused genes along with 5

housekeeping (reference) genes. In addition, they contain a panel of
patented controls to monitor genomic DNA contamination, strand cDNA Run in your real-time
PCR instrument
synthesis and real-time PCR efficiency.
Delta Rn

Why use RT2 Profiler and lncRNA PCR Arrays?

1.E+001

1.E-000

1.E-001

• Content: Select from over 180 different pathways, diseases or bio- 1.E-002

1.E-003
0 4 8 12 16 20 24 28 32 36 40

logical processes and Cycle number

perform routine gene expression analysis on any qPCR instrument

Data analysis
• Control: The integrated controls allow the comparison of results
results from start to finish for consistency

• Customization: Easily modify catalog arrays to fit your needs by p-Value for fold change
10-6

10-5
Normal breast
1

10-1

adding up to 4 genes to make a Modified RT PCR Array; or use 2 10-4

10-3
10-2

10-3

10-4

your gene list to make a full Custom RT2 PCR Array

10-2
10-5
10-1

10-6
1 10-6 10-5 10-4 10-3 10-2 10-1 1
-4 -3 -2 -1 0 1 2 3 4
Breast tumor

From screening canonical pathways to validation of RNA sequencing –

Fold change ratio [log2]

there is always the right RT2 PCR Array for you! Figure 4. The workflow for the RT2 PCR Arrays.

p value
10–9 Figure 5. The Common Cytokine RT2 Profiler PCR Array
10–8 IL10 IL9
IL1A IFNG CSF2 identifies 23 upregulated and 6 downregulated genes
TNF IL2
10–7 IL1B IL11 following PBMC stimulation. Total RNA samples from human
TNFSF10 LTA IL22
CSF1 IL21 IL3
10–6 IL5 IL13 peripheral blood mononuclear cells (either untreated or
10–5 TNFSF13B TGFB2 PDGFA stimulated with 50 ng/ml PMA and 1 mg/ml ionomycin for
TNFSF11 IL17
10–4 6 hours) were characterized in triplicate using the human
IL1F7 BMP6 TNFRSPSF11B
10–3 IFNA5 TNFSF14 Common Cytokine RT2 Profiler PCR Array. Results show
10–2 BMP3 upregulation of 23 genes (>5-fold, p<0.0005) including
10–1 interleukins, colony-stimulating factors and TNF ligands and
100 downregulation of 6 interleukin and TNF ligand genes.
–7 –5 –3 –1 1 3 5 7 9 11 13 15 17
Fold difference (log2)

Technical Guide to QIAGEN PCR Arrays 06/2016  5

 01 02 03 04 05 06 07 08 09 10 11 12

13 14 15 16 17 18 19 20 21 22 23 24

25 26 27 28 29 30 31 32 33 34 35 36
RT2 37
PCR38Array
39 plate
40 41 layouts
42 43and44controls
45 46 47 48

Patented
49 Controls
50 51standardize
52 53qPCR
54 performance
55 56 57 58 59 60
Multiple reference or housekeeping genes (RF or HK) and patented controls (US patent #8,597,938)
Reference gen
61 of62
are a feature every 63
catalog64
RT2 PCR65 (Figures67
Array 66 6 and 68 69 70
7). The inclusion 71 reference
of multiple 72
genes enables researchers to choose the best way to normalize their qPCR experiments. Genomic DNA
73 74 75 76 77 78 79 80 81 82 83 84
Reverse transc
RF RF RF RF RF GDC RTC RTC RTC PPC PPC PPC
Positive PCR c
Figure 6. Key to the controls in the RT2 PCR Array plates. The genomic DNA control (GDC) assay is a sensitive assay that detects
the presence of genomic DNA. The reverse transcription control (RTC) assay detects an artificial RNA template provided with
the RT2 First Strand Kit. The positive PCR controls (PPC) monitor for PCR inhibitors. During data analysis, the online software
monitors ratios between the PPC and RTC to calculate the reverse transcription efficiency. The software also looks at both
variability in a single sample and across samples by monitoring the reproducibility of the RTC and PPC assays.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A 01 01 02 02 03 03 04 04 05 05 06 06 07 07 08 08 09 09 10 10 11 11 12 12

01 02 03 04 05 06 07 08 09 10 11 12 B 01 01 02 02 03 03 04 04 05 05 06 06 07 07 08 08 09 09 10 10 11 11 12 12

C 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24

13 14 15 16 17 18 19 20 21 22 23 24 D 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24

E 25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 35 35 36 36

25 26 27 28 29 30 31 32 33 34 35 36 F 25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 35 35 36 36

G 37 37 38 38 39 39 40 40 41 41 42 42 43 43 44 44 45 45 46 46 47 47 48 48

37 38 39 40 41 42 43 44 45 46 47 48 H 37 37 38 38 39 39 40 40 41 41 42 42 43 43 44 44 45 45 46 46 47 47 48 48

49 50 51 52 53 54 55 56 57 58 59 60
I
J
Gene-specific assays
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K Housekeeping genes
61 61 62 62 63 63 64 64 65 65 66 66 67 67 68 68 69 69 70 70 71 71 72 72
61 62 63 64 65 66 67 68 69 70 71 72 L 61 61 62 62 63 63 64 64 65 65 66 66 67 67 68 68 69 69 70 70 71 71 72 72

M Genomic DNA control

73 73 74 74 75 75 76 76 77 77 78 78 79 79 80 80 81 81 82 82 83 83 84 84
73 74 75 76 77 78 79 80 81 82 83 84
N
Reverse transcription controls
73 73 74 74 75 75 76 76 77 77 78 78 79 79 80 80 81 81 82 82 83 83 84 84

O RFG RFG RFG RFG RFG RFG RFG RFG RFG RFG GDC GDC RTC RTC RTC RTC RTC RTC PPC PPC PPC PPC PPC PPC
HK1 HK2 HK3 HK4 HK5 GDC RTC RTC RTC PPC PPC PPC
P Positive PCR controls
RFG RFG RFG RFG RFG RFG RFG RFG RFG RFG GDC GDC RTC RTC RTC RTC RTC RTC PPC PPC PPC PPC PPC PPC

Reference genes Genomic DNA control

Catalog RT2 PCR Array plate layout for Catalog
ReverseRT 2
PCR Array
transcription controls plate layout
Positive for
PCR controls

96-well qPCR instruments. 384-well qPCR instruments.

1 sample per plate = 96 qPCR assays per sample 4 samples per plate = 96 qPCR assays per sample

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Well 1
A 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Empty
B 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

C 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72
PPC
D 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 RTC
E 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 GDC
F 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 RF
G 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168

H 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192
Postive PCR control
I 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216
Reverse transcription
J 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240
control
K 241 242 243 244 245 246 247 248 249 250 251 253 254 255 256 257 258 259 260 261 262 263 264 265 Genomic DNA control
L 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 Reference genes
M 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312

N 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336

O 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360

P 361 362 363 364 365 366 367 368 369 370 371 372 373 374 Ref1 Ref2 Ref3 Ref4 Ref5 GDC RTC RTC PPC PPC

Reference genes Genomic DNA control

Catalog
ReverseHigh-content RT2 PCR
transcription controls PositiveArray plate layout
PCR controls Catalog RT2 PCR Array plate layout for
for 384-well qPCR instruments. 100-well rotary qPCR instruments.
1 sample per plate = 384 qPCR assays per sample 1 sample per plate = 96 qPCR assays per sample

Figure 7. 96- and and 384-well plate and 100-well ring layouts. The key to the controls is in Figure 6.

6  Technical Guide to QIAGEN PCR Arrays 06/2016

 1. Convert miRNA to cDNA in a one-step, single-tube
miRNA expression profiling using miScript miRNA reverse transcription reaction.
PCR Arrays
miScript miRNA PCR Arrays are mature miRNA-specific forward primers
that have been arrayed in miRNome and biologically relevant, pathway- RNA sample 1 RNA sample 2

focused panels (Table 1). These PCR arrays are provided in ready-to-use,
96- and 384-well plate and 100-well Rotor-Disc® formats. miScript miRNA miRNA
5' 3' Polyadenylation
PCR Arrays are available for various species, provide guaranteed
Poly(A) tail
high performance and are fully customizable. Each array contains 5' 3'
A A A A A(A)n
controls that allow monitoring of the complete experiment from sample
Reverse
preparation to data analysis, including data normalization controls, 5' 3'
transcription
Single-tube reaction A A A A A(A)n
reverse transcription controls and PCR controls. Every assay in a miScript TTT T TT

Universal RT primer
miRNA PCR Array has been bench validated to ensure sensitive and
specific detection of mature miRNA via real-time PCR. First strand cDNA TTT T TT
5' 3'

Table 1. The available miScript miRNA PCR Arrays

Array Species 2. Combine cDNA with QuantiTect SYBR Green PCR

Complete miRNome Human, mouse, rat, dog, rhesus macaque Mastermix, miScript Universal Primer, and water.
Aliquot mixture across miScript miRNA PCR Array.
miFinder Human, mouse, rat, dog, rhesus macaque

Brain Cancer Human, mouse, rat

miScript Primer Assay
Breast Cancer Human, mouse, rat TTT T TT
5' 3'
Cancer PathwayFinder Human, mouse, rat cDNA
miScript Universal Primer
Cell Differentiation & Development Human, mouse, rat

Immunopathology Human, mouse, rat

Inflammatory Response & Autoimmunity Human, mouse, rat

Neurological Development & Disease Human, mouse, rat qPCR using miScript
Primer Assay and
Ovarian Cancer Human, mouse, rat miScript Universal Primer

Serum & Plasma Human, mouse, rat 3. Run in real-time PCR cycler.
Custom Array Human, mouse, rat, dog, rhesus
macaque, and other species

4. Analyze data.

A straightforward, rapid workflow

Sample 2

miRNA expression profiling with miScript miRNA PCR Arrays is simple 1

10-1

and robust (Figure 8). cDNA preparation with the miScript II RT Kit using 10-2

miScript HiSpec Bufferis followed by the addition of a premix of cDNA, 10-3

10-4
miScript Universal Primer, QuantiTect® SYBR Green PCR Master Mix and 10-5

RNase-free water to a miScript miRNA PCR Array. The reaction is run in 10-6
10-6 10-5 10-4 10-3 10-2 10-1 1
Sample 1
a real-time PCR cycler and data analysis is performed using the miScript
miRNA PCR Array Data Analysis Tool. Figure 8. The workflow for the miScript miRNA PCR Arrays.

Technical Guide to QIAGEN PCR Arrays 06/2016  7

PCR Arrays by Application

Research area Apoptosis research Biomarker research Cancer research Cell cycle research

Array type Process, pathway or cell type for analysis

RT2 Profiler PCR Arrays

Apoptosis Alzheimer’s disease Angiogenesis Apoptosis

Autophagy Angiogenesis Apoptosis Autophagy

Breast cancer and estrogen Breast cancer and estrogen

Cancer pathways Cancer pathways
receptor signaling receptor signaling

Cancer drug resistance and

Cell cycle Cancer pathways Cell cycle
metabolism

DNA damage signaling DNA damage signaling

Cell surface markers Cancer pathways
pathway pathway

Dendritic and antigen

DNA repair Cell cycle DNA repair
presenting cell

Epigenetic chromatin modifica- DNA Damage Signaling Epithelial to mesenchymal

Endothelial cell biology
tion enzymes Pathway transition (EMT)

Epigenetic chromatin remodel-

Heat shock proteins EGF/PDGF signaling pathway MAP kinase signaling pathway
ing factors

Epithelial to mesenchymal Epithelial to mesenchymal

NFκB signaling pathway mTOR signaling
transition (EMT) transition (EMT)

Oxidative stress and Extracellular matrix and Neurogenesis and neural

MAP kinase signaling pathway
antioxidant defense adhesion molecules stem cell

p53 signaling pathway Glucose metabolism p53 Signaling Pathway NFκB Signaling Pathway

Hematopoietic stem cells and

PI3K-AKT signaling pathway PI3K-AKT signaling pathway p53 signaling pathway
hematopoiesis

RT2 lncRNA PCR Array

Apoptosis lncRNAs lncRNA biomarkers Cancer lncRNAs Cell cycle regulatory lncRNAs

miScript miRNA PCR Array

Apoptosis Serum & plasma Tumor suppressor Cell cycle regulatory miRNAs

Cell differentiation &

Common miRNAs Tumor suppressor Cancer stem cells
development

Inflammatory response &

Tumor suppressor Cancer regulatory miRNAs Tumor suppressor miRNAs
autoimmunity

miRNome miRBase version 21

8  Technical Guide to QIAGEN PCR Arrays 06/2016

 Signal transduction Stem cell Toxicology/drug
Inflammation research ECM/adhesion research Neuroscience research research research ADME research

Process, pathway or cell type for analysis

Angiogenic growth
Chemokines and cAMP/Ca2+ signaling Cancer drug resistance
factors and Alzheimer’s disease Adipogenesis
receptors pathways and metabolism
angiogenesis inhibitors

EGF/PDGF Signaling Dendritic and antigen-

Common cytokines Atherosclerosis Apoptosis Cancer pathways
pathway presenting cell

Chemokines and G protein-coupled

Inflammasomes Autophagy Embryonic stem cells Cardiotoxicity
receptors receptors

Inflammatory cytokines Hedgehog signaling

Common cytokine Drug transporters GPCR signaling pathways Cell cycle
and receptors pathway

Inflammatory response Hematopoietic stem cells DNA damage signaling

Embryonic stem cells Embryonic stem cells Heat shock proteins
and autoimmunity and hematopoiesis pathway

Hedgehog signaling
Interferon α, β response Endothelial cell biology GPCR signaling pathways Homeobox (HOX) genes Drug metabolism
pathway

Extracellular matrix and Lipoprotein signaling and Drug metabolism: phase I

Interferon and receptor Heat shock proteins Insulin signaling pathway
adhesion molecules cholesterol metabolism enzymes

JAK/STAT signaling Hedgehog signaling JAK/STAT signaling Drug metabolism: phase

Glycosylation Mesenchymal stem cell
pathway pathway pathway II enzymes

MAP kinase signaling MAP kinase signaling Neurogenesis and neural

NFκB signaling pathway Huntington’s disease Drug transporters
pathway pathway stem cell

T Cell energy and Hypoxia signaling Neurotrophin and

Mesenchymal stem cell mTOR signaling GPCR signaling pathway
immune tolerance pathway receptors

T-cell and B-cell

NFκB signaling pathway Mesenchymal stem cell NFκB signaling pathway Notch signaling pathway Hepatoxicology
activation

TGFβ BMP signaling Neurogenesis and neural Nuclear receptors and Lipoprotein signaling and
Osteogenesis Osteogenesis
pathway stem cell coregulators cholesterol metabolism

Inflammation response ECM & adhesion Signal transduction Development & Common lncRNA
Neuroscience lncRNAs
lncRNAs lncRNAs lncRNAs differentiation lncRNAs biomarkers

Neurological develop-
T-cell & B-cell activation Fibrosis Hypoxia signaling Stem cells Serum & plasma
ment & disease

Cell differentiation & Neuropathic & inflamma- Cell differentiation &

Immunopathology Common miRNAs Liver miRNAs
development tory pain development

Inflammatory response &

Apoptosis Serum & plasma Apoptosis Cancer stem cells Cardiovascular miRNAs
autoimmunity

miRNome miRBase version 21

Technical Guide to QIAGEN PCR Arrays 06/2016  9

Discover biomarkers in serum or plasma Empty
Well 1

PPC
The range of pathway-focused miScript miRNA PCR Arrays is continually miRTC
expanding to enable new discoveries about the roles of miRNAs in biological snoRNA
miRNA
processes. Content is selected using our proprietary methodology, which
C. elegans
ensures that the arrays are up-to-date and biologically relevant. Postive PCR control
miRTC miScript Primer Assay
miScript Primer Assays
One of the most exciting areas of current miRNA research involves the C. elegans miScript Primer Assay
snoRNA miScript PCR Controls
assessment of miRNAs present in serum or plasma samples. The relatively
stable, extracellular miRNAs in serum and plasma have great potential
as biomarkers for a variety of diseases. Some could find use in liquid
biopsies – a minimally invasive way of assessing disease states.

The Serum & Plasma miScript miRNA PCR Array has been developed to
01 02 03 04 05 06 07 08 09 10 11 12
enable rapid profiling of the 84 most relevant disease-associated miRNAs 01 02 03 04 05 06 07 08 09 10 11 12

13 and
in serum 14 plasma,
15 16 17 18
supporting liquid19 20 research.
biopsy 21 22 23 24 13 14 15 16 17 18 19 20 21 22 23 24

25 26 27 28 29 30 31 32 33 34 35 36
25 26 27 28 29 30 31 32 33 34 35 36
miScript miRNA PCR Array layout 37 38 39 40 41 42 43 44 45 46 47 48

Our 37
arrays38 39 miScript
contain 40 41 Primer 43 for
42Assays 4484 to
4537246miRNAs
47 along
48 49 50 51 52 53 54 55 56 57 58 59 60

61 62 63 64 65 66 67 68 69 70 71 72
with controls for data normalization, reverse transcription and PCR.
49 50 51 52 53 54 55 56 57 58 59 60 73 74 75 76 77 78 79 80 81 82 83 84
The formats are shown in Figure 9. The key to the controls is shown in
Ce Ce SN1 SN2 SN3 SN4 SN5 SN6 miRTC miRTC PPC PPC
6110 62
Figure 63 64 details
with additional 65 66 67 2. 68 69 70 71 72
in Table
73 74 75 76 77 78 79 80 81 82 83 84 Gene-specific assays Reverse transcription controls
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A 01C.
01 elegans
02 02 03 03miR-39
04 04 05 05 06 06 Positive PCR
07 07 08 08 controls
09 09 10 10 11 11 12 12

B
miScript Primer Assay
01 01 02 02 03 03 04 04 05 05 06 06 07 07 08 08 09 09 10 10 11 11 12 12

Ce Ce SN1 SN2 SN3 SN4 SN5 SN6 miRTC miRTC PPC PPC C 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24

D 13 snoRNA/snRNA
13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24

E 25 miScript PCR
25 26 26 27 Controls
27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 35 35 36 36

F 25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 35 35 36 36

Figure 10. Key to the controls for qPCR performance in the miScript miRNA PCR Array plates. G 37 37 38 38 39 39 40 40 41 41 42 42 43 43 44 44 45 45 46 46 47 47 48 48

Gene-specific assays Reverse transcription controls

Data normalization controls include 6 miScript PCR Controls (SN1–6) and a spike-in control H
I
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for monitoring microRNA isolation (C. elegans miR-39; Ce). The Ce assay detects the J

C. elegans miR-39
49 49 50 50 51 51 52 52 53 53 54 54 55 55 56 56 57 57 58 58 59 59 60 60

Positive PCR controls

Syn-cel-miR-39 miScript miRNA Mimic. The reverse transcription control (miRTC) assay K
L
61 61 62 62 63 63 64 64 65 65 66 66 67 67 68 68 69 69 70 70 71 71 72 72

miScript Primer Assay

61 61 62 62 63 63 64 64 65 65 66 66 67 67 68 68 69 69 70 70 71 71 72 72
detects an artificial RNA template provided with the arrays. The positive PCR controls (PPC) M 73 73 74 74 75 75 76 76 77 77 78 78 79 79 80 80 81 81 82 82 83 83 84 84

monitor for PCR inhibitors. N 73 73 74 74 75 75 76 76 77 77 78 78 79 79 80 80 81 81 82 82 83 83 84 84

snoRNA/snRNA
O Ce Ce Ce Ce SN1 SN1 SN2 SN2 SN3 SN3 SN4 SN4 SN5 SN5 SN6 SN6 miRTC miRTC miRTC miRTC PPC PPC PPC PPC

P Ce Ce Ce Ce SN1 SN1 SN2 SN2 SN3 SN3 SN4 SN4 SN5 SN5 SN6 SN6 miRTC miRTC miRTC miRTC PPC PPC PPC PPC

miScript PCR Controls Reference genes Genomic DNA control

Table 2. The controls for qPCR performance in the miScript miRNA PCR Array plates 1
Reverse transcription controls Positive PCR controls
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

B 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Control Purpose C 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72

D 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
C. elegans miR-39 miScript Primer Assay (Ce) Alternative data normalization using E 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

exogenously spiked Syn-cel-miR-39 F 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144

miScript miRNA Mimic G 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168

H 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192

6 snoRNA/snRNA Primer Assays (miScript Data normalization using the ∆∆CT I 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216

PCR Controls; SN1–6) method of relative quantification J 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240

K 241 242 243 244 245 246 247 248 249 250 251 253 254 255 256 257 258 259 260 261 262 263 264 265

L
miRNA reverse transcription control (miRTC) Assessment of reverse transcription 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288

M 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312
performance N 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336

O 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360
Positive PCR control (PPC) Assessment of PCR performance P 361 362 363 364 365 366 367 368 369 370 371 372 Ce Ce SN1 SN2 SN3 SN4 SN5 SN6 miRTC miRTC PPC PPC

Reference genes Genomic DNA control

Figure 9. The
Reverse layouts
transcription of thePositive
controls miScript miRNA PCR Array plates:
PCR controls

the 100-well ring; 96-well plate, and 384-well plates set up

for four samples or 1 sample. Control key in Figure 10.

10  Technical Guide to QIAGEN PCR Arrays 06/2016

Modified and Custom PCR Arrays
01 02 03 04 05 06 07 08 09 10 11 12

13 14 15 16 17 18 19 20 21 22 23 24
Modified PCR Arrays 25 26 27 28 29 30 31 32 33 34 35 36

Add any 4 genes back to a cataloged array to instantly create an 37 38 39 40 41 42 43 44 45 46 47 48

Gene-spec
affordable and unique solution for your research. This is an excellent tool 49 50 51 52 53 54 55 56 57 58 59 60
Housekee
61 62 63 64 65 66 67 68 69 70 71 72
for scaling up your single gene experiments to a pathway, or to verify Genomic
73 74 75 76 77 78 79 80 81 82 83 84
the role of your favorite genes in a biological process. These arrays are Reverse tr
HK1 HK2 HK3 HK4 HK5 GDC RTC 03 01 02 04 PPC
Positive PC
available for RT2 Profiler and RT2 lncRNA PCR Arrays.

96-well format 384-well format

Custom RT2 and miScript PCR Arrays
Custom RT2 and miScript PCR Arrays employ a high-throughput approach
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A

01 02 03 04 05 06 07 08 09 10 11 12 B
C
13 14 15 16 17 18 19 20 21 22 23 24 D

for profiling the expression of genes of interest. Choose any miRNA

E
25 26 27 28 29 30 31 32 33 34 35 36 F
G
37 38 39 40 41 42 43 44 45 46 47 48 H
I

or combine mRNA and lncRNA genes from the supported species and
49 50 51 52 53 54 55 56 57 58 59 60 J
K
61 62 63 64 65 66 67 68 69 70 71 72 L
M
73 74 75 76 77 78 79 80 81 82 83 84 N

chose from several different plate layouts (Figures 11 and 12). Whether
O
Ce Ce SN1 SN2 SN3 SN4 SN5 SN6 miRTC miRTC PPC PPC
P

12 genes – 8 samples/plate 16 genes – 24 samples/plate

your interests are in biomarker discovery, drug development, disease
A
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
characterization, signal transduction or RNA-seq and microarray
B

followup, the Custom RT2 and miScript PCR Arrays enable superior qPCR
C
D
E
F
G

performance without the need for primer validation or optimization.

H
I
J
K
L
M
N

Each qPCR assay used in a custom array goes through the same
O
P

24 genes – 4 samples/plate 32 genes – 12 samples/plate

laboratory-verification process as our standard catalog arrays.

Custom RT2 and miScript PCR Array are delivered within 3 weeks of
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A
B
C
D

ordering and are available in 96- and 384-well plates and 100-ring disc
E
F
G
H
I

formats suitable for QIAGEN Rotor-Gene® Q instruments.

J
K
L
M
N
O
P

Start building your arrays today at qiagen.com/myPCRarray.

12 genes – 3 samples/plate 48 genes – 8 samples/plate

100-well disc format

48 genes – 2 samples/plate 96 genes – 4 samples/plate

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A

01 02 03 04 05 06 07 08 09 10 11 12 B
C
13 14 15 16 17 18 19 20 21 22 23 24 D
E
25 26 27 28 29 30 31 32 33 34 35 36 F
G
37 38 39 40 41 42 43 44 45 46 47 48 H
I
49 50 51 52 53 54 55 56 57 58 59 60 J
K
61 62 63 64 65 66 67 68 69 70 71 72 L
M
73 74 75 76 77 78 79 80 81 82 83 84 N
O
Ce Ce SN1 SN2 SN3 SN4 SN5 SN6 miRTC miRTC PPC PPC
P

96 genes – 1 sample/plate 384 genes – 1 sample/plate 12 genes – 8 samples/plate 96 genes – 1 sample/plate

Figure 11. The layouts of Custom RT PCR Array plates. 2

Figure 12. The layouts of the Custom RT PCR Array Ring.
2

Technical Guide to QIAGEN PCR Arrays 06/2016  11

Full support for data analysis p value
10–9
GeneGlobe RT and miScript PCR Array Data
2 10–8
10–7
Analysis Center 10–6
10–5
This integrated web-based software package for the RT2 and miScript
10–4
PCR Array systems automatically performs all ∆∆CT-based fold-change 10–3
10–2
calculations from your uploaded raw data. Simply providing the array
10–1
catalog number(s) annotates the results to the correct gene list. 100
–7 –5 –3 –1 1 3 5 7
The web portal delivers results not only in a tabular format but also Fold difference (log2)

scatter, volcano, cluster-gram and multi-group plots (Figure 13). Perform

any pair-wise comparison between groups of experimental replicates
by defining your own fold-change and statistical significance thresholds, Breast tumor
101
or compare all of the groups side-by-side. The web portal also helps to
100
correctly interpret genomic DNA, reverse transcription efficiency and
positive PCR control well data. 10–1

10–2
Make your pathway-focused gene expression analysis quick and
10–3
painless with by using the GeneGlobe Data Analysis Portal:
10–4
• Simple: Just upload your data and define your parameters
10–5
• Convenient: No downloading or installation required 10–5 10–4 10–3 10–2 10–1 100 101
Normal breast
• Publication-ready: Export all results as free Excel® files or
Figure 13. Example output plots from the GeneGlobe Data
.png image files Analysis Center. A The volcano plot shows the fold change
and statistical significance of gene expression changes.
Relevent gene expression changes can be ranked by the
p-value allowing researchers to follow-up on highly
consistent fold change values. This becomes very important
when trying to determine if genes with small fold changes
are relevent for future experiments. B The scatter plot
indicated the fold difference of gene expression changes.
Two samples can quickly be compared using this plot.

Instructions
1. Upload your data in a simple Excel file format.

2. Define your experimental groups and replicates.

3. Select your normalization genes or have them automatically selected

for you.

4. Export your data analysis results and graphs.

Test it with a pre-loaded sample data set at qiagen.com/geneglobe/

dataanalysis.

12  Technical Guide to QIAGEN PCR Arrays 06/2016

 GeneGlobe makes your sample to insight research quest easier!

Interested in a specific research area,

biology or species?

Browse by Browse by
research area Browse by species biology

Want access to
qPCR arrays,
scientific literature, Need to analyze
assays,
publications, and interpret your
NGS panels,
webinars and white results?
custom products
papers?

Data Analysis
Center

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recommendations
for your next project!

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GeneGlobe lists Product Finder Next
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Technical Guide to QIAGEN PCR Arrays 06/2016  13

 For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN
Technical Services or your local distributor.

Trademarks: QIAGEN®, Sample to Insight®, QIAzol®, GeneGlobe®, Ingenuity®, miScript®, RNeasy®, Rotor-Disc®, Rotor-Gene® (QIAGEN Group); Excel® (Microsoft Corporation).
© 2016 QIAGEN, all rights reserved. PROM-9494-001

Ordering www.qiagen.com/shop Technical Support support.qiagen.com Website www.qiagen.com

1099972 06/2016