MiRCURY LNA PCR System Manual

7/28/2019 MiRCURY LNA PCR System Manual

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miRCURY LNA Universal RTmicroRNA PCR

Expression

Analysis

Instruction manual v.5.2#203301 March 2013

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miRCURY LNA Universal RT microRNA PCR Instruction Manual

Table of contents

Product Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

I. Reagent kits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

II. Primer Sets and Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Additional required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Recommended accompanying products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Control Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Before starting the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

A.Individual assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

B.Human and Mouse&Rat microRNA PCR Panels. . . . . . . . . . . . . . . . . . . . . . . . . . . 29

C.Focus microRNA PCR Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

D.Pick-&-Mix microRNA PCR Panels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Tips to protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Troubleshooting guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

FAQs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Related products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63


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Product summary

The miRCURY LNA Universal RT microRNA PCR system is a microRNA-specific,

LNA-based system designed for sensitive and accurate detection of microRNA by quantitative

real-time PCR using SYBR Green. The method is based on universal reverse transcription

(RT) followed by real-time PCR amplification with LNA enhanced primers (for more details

please see page 12). The miRCURY LNA Universal RT microRNA PCR portfolio is comprised

of four types of reagent kits; including:

Universal cDNA synthesis kit II

RNA Spike-in kit

ExiLENT SYBR

Green master mix kit microRNA primer sets available in pre-defined Human, Mouse&Rat and Focus PCR panels and

customized Pick-&-Mix PCR panels as well as individual primer sets and reference genes. All

PCR panels are Ready-to-Use delivered with one 10 L PCR reaction per well.

Figure 1.

Universal cDNA synthesis kit II

Description page 4

RNA Spike-in kit

(optional)

+

microRNA and referencegene primer sets

Description page 6,protocol page 20

Description page 9,protocol page 43

Description page 7,protocol page 29+36

Pick-&-Mix PCR panels(Ready-to-Use)

+ExiLENT SYBR Green master mix

2,5 or 20ml

Predefined PCR panelsHuman, Mouse&Rat, and Focus PCR panels

(Ready-to-Use)

Description page 5


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I. Reagent kits

Universal cDNA synthesis kit II, 8-64 rxns (product # 203301)

This kit contains all reagents required for first-strand cDNA synthesis for 8-64 reactions1). The

UniSp6 RNA Spike-in template can be used by itself or in combination with the cel-miR-39-3p

template provided in the RNA Spike-in kit:

Contents

5 x Reaction buffer2) 128 L, 5 x concentrated

Enzyme mix 64 L, 10 x concentrated

Nuclease free water 1 ml

UniSp6, RNA Spike-in template 12 fmol, dried down

1) Number of reactions is based on a standard reaction volume of 10 L to 80 L. Reaction volume depends on the application

and number of assays to profile. Please consult Figure 4 for details.2) Includes universal reverse transcr iption primer.3) Used exclusively for the UniSp6 RNA Spike-in control primer set included in the ExiLENT SYBR Green master mix kit.


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ExiLENT SYBR Green master mix, 2.5 ml (product# 203402)

This kit contains all reagents required for PCR amplification of microRNAs. In addition, a

positive control assay, the UniSp6 RNA Spike-in control primer set, is provided with this kit for

amplification of the synthetic UniSp6 RNA spike-in provided in the Universal cDNA synthesis

kit II. The reagents are provided in amounts sufficient for 500 reactions of 10 L.

ExiLENT SYBR Green master mix, 20 ml (product# 203420)

This kit contains all reagents required for PCR amplification of microRNAs. In addition, a

positive control assay, the UniSp6 RNA Spike-in control primer set, is provided with this kit for

amplification of the synthetic UniSp6 RNA spike-in provided in the Universal cDNA synthesis

kit II (the UniSp6 RNA Spike-in control primer set is provided 100x concentration and should

be diluted to 10x concentration before using in the protocol for individual assays). The reagentsare provided in amounts sufficient for 4000 reactions of 10 L.

Contents

PCR Master mix 2 x 1.25 ml, 2x concentrated

Nuclease free water 2 x 1.25 ml

UniSp6 RNA Spike-in control primer set1) 500 L, 10x concentrated

Contents

PCR Master mix 2 x 10 ml, 2x concentrated

Nuclease free water 1 x 20 ml

UniSp6 RNA Spike-in control primer set1) 500 L, 100x concentrated

1) Used exclusively for detection of the UniSp6 RNA spike-in provided with the Universal cDNA synthesis kit II.


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II. Primer Sets and Panels

MicroRNA LNA PCR primer set (product# 204000-205xxx)

LNA PCR primer sets are designed for optimal performance with the Universal cDNA

Synthesis Kit II and the ExiLENT SYBR Green master mix, kit II. The performance of LNA

primer sets will be affected if used in combination with less than optimal reagents. The primer

sets are supplied in sufficient amounts for 200 reactions of 10 L.

Reference gene primer set (product # varies)

The Reference gene primer sets are designed for use with the microRNA primers sets above

as reference genes for normalization. The primer mix is supplied in sufficient amount for 200

reactions of 10 L.

Contents

LNA PCR Primer mix (dried down)

ContentsLNA PCR Primer mix (dried down)


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Pre-defined microRNA PCR Panels (product # varies)

The Human, Mouse&Rat and Focus microRNA PCR panels Ready-to-Use consist of either

96-well or 384-well PCR plates containing dried down LNA primer sets for one 10 L real-

time PCR reaction per well. The LNA primer sets are designed for optimal performance

with the Universal cDNA Synthesis Kit II and the ExiLENT SYBR Green master mix, kit. The

performance of the LNA primer sets will be affected if they are used in combination with

less than optimal reagents.

Contents Human miRNome panel I and II, V3384-well PCR plates supplied with LNA primer sets dried down, one 10 L reaction per well:

Contents Mouse&Rat miRNome panel I and II, V3

384-well PCR plates supplied with LNA primer sets dried down, one 10 L reaction per well:

Panel I Panel II

372 LNA primer sets for theamplification of human microRNAs1)

380 LNA primer sets for theamplification of human microRNAs1)

3 inter-plate calibrators 3 inter-plate calibrators

3 primer sets for reference genes2) 1 blank well

5 RNA Spike-in control primer sets 3)

1 blank well

Panel I Panel II

372 LNA primer sets for the

amplification of mouse and rat microRNAs1)

380 LNA primer sets for the

amplification of mouse and rat microRNAs1)

3 inter-plate calibrators 3 inter-plate calibrators

3 primer sets for reference genes2) 1 blank well

5 RNA Spike-in control primer sets 3)

1 blank well


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Contents Serum/Plasma Focus microRNA PCR panel, V3

PCR plates compatible with various real-time PCR instruments are available and supplied

with LNA primer sets dried down, one 10 L reaction per well:

Contents Cancer Focus microRNA PCR panel, V3

PCR plates compatible with various real-time PCR instruments are available and supplied

with LNA primer sets dried down, one 10 L reaction per well:

96-well (2 plates) 384-well plate (2 panels per plate)

179 LNA primer sets for the amplificationof human microRNAs1+2)

2x179 LNA primer sets for the amplification of humanmicroRNAs1+2)

2x3 Inter-plate calibrators 2x6 Inter-plate calibrators

5 RNA Spike-in control primer sets 3) 2x5 RNA Spike-in control primer sets3)

2 blank wells - 1 in each plate 2x2 blank wells

96-well (1 plates) 384-well plate (4 panels per plate)

84 LNA primer sets for the amplificationof human microRNAs1)

4x84 LNA primer sets for the amplification of humanmicroRNAs1)

3 Primer sets for potentialreference genes2)

4x3 Primer sets for potentialreference genes2)

3 Inter-plate calibrators 4x3 Inter-plate calibrators

5 RNA Spike-in control primer sets 3) 4x5 RNA Spike-in control primer sets 3)

1 blank well 1 blank well

1) Please go to www.exiqon.com/mirna-pcr to download plate layout files.2) Human Panels and Cancer Focus Panel: Three snRNAs (U6snRNA, SNORD38B, SNORD49A). Mouse&Rat Panels: Three

snRNAs (U6snRNA, RNU5G, RNU1A1). Serum/plasma Focus Panel: miR-103, miR-191, miR-423-5p, miR-16, miR-425,

miR-93, miR-451 are regarded reference gene candidates.3) The RNA Spike-in control primer sets targets the UniSp6 RNA spike-in supplied in the Universal cDNA synthesis kit II

and the 4 RNA spike-ins contained in the RNA Spike-in kit (UniSp2, UniSp4, UniSp5, and cel-miR-39-3p).


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Pick-&-Mix microRNA PCR Panel, (product # 203801 and 203802)

The Pick-&-Mix microRNA PCR Panels consist of either 96-well PCR plates or 384-well

PCR plates containing custom selections of dried down microRNA LNA PCR primer sets

for one 10 L real-time PCR reaction per well, ready-to-use. PCR plates compatible with

various real-time PCR instruments are available. The LNA primer sets are designed for

optimal performance with the Universal cDNA Synthesis Kit II and the ExiLENT SYBR Green

master mix kit. The performance of the LNA primer sets will be affected if they are used in

combination with less than optimal reagents.

Contents

PCR plates supplied with customer defined LNA primer sets, reference gene primer sets,

and RNA Spike-in control primer sets, dried down, one 10 L reaction per well1):

96-well PCR plates1) 384-well PCR plates1)

10 primer sets in 8 replicates 22 primer sets in 16 replicates

22 primer sets in 4 replicates 46 primer sets in 8 replicates

92 primer sets in 1 replicate 94 primer sets in 4 replicates

96-well flexible layout 380 primer sets in 1 replicate

384-well flexible layout

1) Please go to www.exiqon.com/pick-and-mix to configure a Pick-&-Mix microRNA PCR Panel.


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Storage

All microRNA PCR Panels, LNA primer sets and Reference gene primer sets

The PCR panels and primer sets are shipped dried down at room temperature. The primers

can be stored between +4C and -20C. Under these conditions, all components are stable

for at least 12 months. After resuspension, it is recommended to store LNA primer sets

and Reference gene primer sets in aliquots at -20C to avoid repeated freeze-thaw cycles.

Universal cDNA synthesis kit II and ExiLENT SYBR

Green master mixThese kits are shipped on dry ice in polystyrene containers and should be stored at -15C to

-25C. Do not store in a frost-free freezer. Under these conditions, all components are stable

until the expiry date on the package or vial. It is recommended that the RNA spike-in be stored

in aliquots at -20C after re-suspension to avoid repeated freeze-thaw cycles.

Additional required materials

Reagents not supplied

ROX or other passive reference dye (required on some PCR cyclers)

Materials and Equipment not supplied

Nuclease-free PCR tubes or plates for use with individual assays

Nuclease-free, aerosol barrier pipette tips

Nuclease-free, low nucleic acid binding microcentrifuge tubes

(e.g. Eppendorf DNA LoBind tubes product and original NUNC vials)

Sealing foils for PCR plates

Micro-centrifuge and plate centrifuge

Heating block, thermal cycler or other incubators

Real-time PCR instrument

Optional but recommended product

miRCURY LNA Universal RT microRNA PCR, RNA Spike-in kit

(product# 203203)


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Recommended accompanying products

Exiqon recommends the Exiqon GenEx qPCR software for comprehensive and convenient data

analysis. GenEx includes a wizard for import of Exiqon miRCURY Universal RT microRNA PCR

data and offers advanced methods to analyze real-time qPCR data in a few simple steps. The

software includes tools for selection and validation of reference genes, data pre-processing and

comprehensive statistical analyses. For more information and to download a free trial, please

go to www.exiqon.com/qpcr-software. The following Exiqon GenEx products are available:

Exiqon GenEx Industrial - Exiqon version of GenEx,

qPCR analysis software, industrial license

Exiqon GenEx Academic - Exiqon version of GenEx,

qPCR analysis software, academic license

Exiqon recommends the miRCURY RNA Isolation kits for purification of total RNA or small

RNA fraction. RNA purified using the miRCURY RNA Isolation kits is fully compatible with

the miRCURY LNA Universal RT microRNA PCR System. The following kits are available:

miRCURY RNA Isolation Kit Cell & Plant

Provides a rapid method for purification of total RNA from cultured animal cells, small tissue

samples, blood, bacteria, yeast, fungi and plants.

miRCURY RNA Isolation Kit Tissue

Specifically designed for purification of total RNA from tissue samples.

miRCURY RNA Isolation Kit Biofluids

Kit for purification of low abundant small RNAs from samples such as serum, plasma, urine

and CSF.

See Tip 2


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Product description

A unique system for microRNA profiling

miRCURY LNA Universal RT microRNA PCR offers the best available combination of

performance and ease-of-use on the microRNA real-time PCR market because it unites two

important features (Figure 2):

1. Universal RT One first-strand cDNA synthesis reaction provides template for all microRNA

real-time PCR assays. This saves precious sample, reduces technical variation, requires use

of less reagents, and saves time in the laboratory.

2. LNA PCR amplification Both PCR amplification primers (forward and reverse) are

microRNA specific and optimized with LNA. The result is: 1) exceptional sensitivity as well as

extremely low background enabling accurate quantification of very low microRNA levels and 2)

highly specific assays that allow discrimination between closely related microRNA sequences.

miRCURY LNA Universal RT microRNA PCR offers solutions both for high-throughput

microRNA expression profiling and for quantification of individual microRNAs.

Step 1: First-strand synthesis (RT)

Step 2: Real-time PCR amplification

Mature microRNAAAAAAAAAAAAAAAAAAAAAA)

3 degenerate anchor

miR-specific forward primer

miR-specific reverse primer

AAAAAAAAAAAAAAAAAAAATTTTTTTTTTTTTTT

TTTTTTTTTTTTTTT

5 universal tag

B)

A)

B)

Figure 2. Schematic outline of the miRCURY LNA Universal RT microRNA PCR System. A poly-A tail is added to the

mature microRNA template (step 1A). cDNA is synthesized using a poly-T primer with a 3 degenerate anchor and a 5

universal tag (step 1B). The cDNA template is then amplified using microRNA-specific and LNA-enhanced forward and

reverse primers (step 2A). SYBR Green is used for detection (step 2B).


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Control Assays

There are 3 different types of control assays available in the miRCURY LNA Universal RT

microRNA PCR system:

Reference assays and reference candidates

Inter-plate calibrators

RNA spike-in assays

All of these control assays are available in the microRNA PCR panels. One RNA spike-in templateis provided with the Universal cDNA Synthesis Kit II, while the assay that will detect this RNA

spike-in is available in the ExiLENT SYBR Green master mix. Additionally 4 RNA spike-in

templates are available as a spike-in kit. The assays for detection of these 4 templates as well

as the reference assays are available as individual primer sets.

Reference assays and reference candidates

These assays detect small non-coding RNAs - either small nuclear RNA, small nucleolar RNA or

microRNA which frequently are found to be stably expressed across different cells or tissues.

Reference assays may therefore be candidate assays for normalization in a profiling study withseveral samples. Though this is a good and recommended approach, great caution should be taken

in the selection of reference genes. The danger of using endogenous reference genes lies in the

assumption that a specific gene is expressed at the exact same level in all sample types. This is

rarely true. The selection of reference genes should therefore be made with care, and should be

specific to the sample set you are working with. The actual selection of reference genes to be used for

normalization should always be based on a determination of the most stably expressed gene(s) which

may be done using either GeNorm or NormFinder both tools that are integrated within Exiqons

GenEx data analysis software. When applicable, we recommend using microRNA rather than small

nuclear RNA or small nucleolar RNA for normalization. Firstly, small nuclear and nucleolar RNAs

are longer RNA species than microRNA and may purify differently from microRNA. Moreover, small

nuclear and nucleolar RNAs have entirely different functions as well as sub-cellular locations, and

finally certain samples like blood plasma does not contain the small nuclear and nucleolar RNAs.

Global mean normalization is a preferred alternative to using reference genes for normalization

when working with panels and samples where many microRNAs are screened per sample and where

many microRNA are called (detected) in all samples. Exiqons GenEx will easily perform global mean

normalization. To read more on normalization.

See Tip 11


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Inter-plate calibrators

Three wells within the pre-defined Human, Mouse&Rat, and Focus PCR Panels contain the

inter-plate calibrator assay (annotated as UniSp3 IPC in the plate layout files). Depending

on the plate layout, the Pick-&-Mix Panels contain at least three inter-plate calibrators.

Each of these wells, contain a pre-aliquoted primer pair and a DNA template and therefore

the variation is very minimal from well-to-well and from plate-to-plate of these assays. The

inter-plate calibrators are used for calibration between PCR plate runs which is very useful

on some instruments that apply the cycle threshold method for Cq determination such as

the ABI7900 PCR cycler. Since the inter-plate calibrators are independent of cDNA quality inorder to give a signal (but may be affected by PCR inhibitors in the sample) they may be used

to quality control each plate run.

Inter-plate calibration (IPC) can easily be performed in the data analysis software Exiqons

GenEx. Alternatively, IPC may be performed manually by using the IPC assay replicates as

follows. For each plate, verify that the replicates have Cq standard deviation within 0.5. If

this is not the case, eliminate the outlier if this can be identified. Calculate the average of

the replicates for each plate, the overall average (average of IPC values from all plates). The

calibration factor is calculated as the difference between plate average and overall averagefor each plate (calibration factor = IPCplate-IPCoverall). An example is shown in Table 1.

Finally, calibrate each plate by subtracting the calibration factor from all Cq values in the plate.

Table 1. Example of inter-plate calibration (IPC)

Plate 1 Plate 2 Plate 3

let-7c 21.12 20.93 21.34

IPC plate average 19.72 19.70 20.00

IPC overall average 19.81 19.81 19.81

Calibration factor -0.09 -0.11 0.19

let-7c calibrated 21.21 21.04 21.15


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RNA spike-ins (synthetic control templates)

The primary purpose of the RNA spike-ins and the matching primer pairs for detection of

these is to provide controls for the quality of the RNA isolation, the cDNA synthesis reaction

and the PCR. RNA isolations may vary in yield, purity and integrity. Some sample types may

contain compounds that inhibit the cDNA synthesis or the PCR even though the RNA has been

purified using the best standard procedures. This may result in different efficiencies of the

reverse transcription or PCR between compared samples. One way to control for differences

in efficiencies at each experimental level (isolation, cDNA synthesis, and PCR) is by adding

known RNA spike-ins to the sample prior to isolation and cDNA synthesis. Use of the RNAspike-ins may also reveal if nucleases are present. After conducting the PCR but before

initiating the data analysis, wells detecting RNA spike-ins are compared and outlier samples

may be identified and considered for exclusion in the further data analysis.

We have designed a flight of RNA spike-ins for this purpose. The UniSp6 RNA Spike-in

template is provided with the Universal cDNA synthesis kit II. Additionally four RNA spike-

in templates can be obtained with the separate RNA Spike-in kit. Here, a vial of three RNA

spike-in templates, UniSp2, UniSp4 and UniSp5, mixed at different concentrations can be

used during RNA isolation. The cel-miR-39-3p RNA template provided in a separate vial in theRNA Spike-in kit can be mixed with the UniSp6 template from the Universal cDNA synthesis

kit II to obtain two different template concentrations. This combination can be added during

the cDNA synthesis. Five wells in pre-defined PCR panel plates contain the matching primer

sets. The selection of RNA Spike-in control primer set in the Pick-&-Mix PCR Panel can be

customized to the specific need. A UniSp6 control primer set is also provided with the ExiLENT

SYBR Green master mix kit, which is to be used with our non-plate based PCR primer set

products.The RNA spike-ins are shipped dried down and must be re-suspended before use.

If the RNA Spike-in kit with multiple RNA spike-ins is used, follow the protocol accompanying

that product.

If UniSp6 is to be used alone:

1. Re-suspend the UniSp6 RNA spike-in by adding 80 L nuclease free water to the tube.

2. Mix by vortexing and spin down. Leave for 20-30 min. on ice to fully dissolve RNA spike-in.

Mix by vortexing and spin down. Store in aliquots at -20C

3. Prior to the RT reaction, add 1 L synthetic spike-in (108 copies/L) per 20 ng sample RNA.


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An alternative application of the UniSp6 RNA spike-in is as inter-plate calibrator. This

is only relevant when using individual LNA primer sets and Reference gene primer

sets in a multi-plate set-up. The microRNA PCR panels already contain an inter-plate

calibrator. Add 1 L synthetic spike-in (108 copies/L) to 20 ng of a complex RNA sample

(e.g. total RNA from MS2, yeast, or a cell line; not provided with the kit). Proceed with first

strand synthesis and subsequently real-time PCR as described in the protocols of the current

instruction manual. At least one spike-in amplification reaction per PCR plate is used for

inter-plate calibration.

Experimental design

Before starting the experiment, it is essential to consider the experimental setup and consider

the number of replicates needed for obtaining significant results replicates being technical

as well as biological. The number of biological replicates required varies from experiment to

experiment depending on the variation within and between the groups. We recommend that a

No Template Control (NTC) is included in the study every time a new experiment is set up, to

set the background level. The most optimal NTC is a mock up sample preparation including

only carrier RNA as sample. The NTC should be run on all assays included in the study. Wedefine the background of an assay as 5 Cq values below the NTC level. Furthermore we

recommend including spike-ins found in the Spike-in kit to provide full quality control over

all steps in the profiling (see figure 3). Finally it is necessary to include a number of candidate

reference miRNAs, which are expected to be constitutively expressed across the different

experimental conditions, for data normalization.

Figure 3. Experimental design

See Tip 9

UniSp2

Sample n

Sample n+1 Sample preparation evaluation

Sold as separate Spike-in kit (203203)

cDNA synthesis evaluation

Comes withUniversal cDNAsynthesis kit II

Included in allpanel products

Assays available asindividual assays or inready-to-use panels

Inter plateCalibration

Sample profiling

NTC

UniSp4 UniSp5 cel-miR-39-3p UniSp6 UniSp3 miR-A miR-X


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Before starting the Experiment

Before setting up a real-time PCR experiment, there are a number of practical experimental

design parameters that should be considered:

RNA input - The miRCURY LNA Universal RT microRNA PCR protocol is optimized for use

of 20 ng total RNA per 20L cDNA synthesis reaction. The exact amount of total RNA needed

depends on whether the downstream application is individual assays or panels. Furthermore,

the amount of total RNA to be used may also vary depending on the microRNA expression

levels in the cells or tissue to be analyzed. For highly expressed microRNAs it is possible to

use down to 10 pg total RNA as starting material. For weakly expressed microRNAs it may

be possible to use up to 200 ng of total RNA; however, in samples with high amounts of PCR

inhibitors (e.g. FFPE tissue samples), this may not be feasible. Finally, inhibitors may be presentin RNA preparations from certain samples e.g. serum and plasma. Prior to conducting a

larger microRNA profiling study, it is recommended to optimize the amount of input RNA to

the RT reaction in order to avoid conducting a larger study where inhibition occurs sporadically

throughout the data set.

Information on how to extract and handle RNA can be found in the tips section. In short, total

RNA should be prepared using a method that preserves small RNA species. DNase treatment

may be necessary. When using commercially available kits, please ensure that the total RNA

preparation is guaranteed to contain microRNAs.

RNA work requires specific handling and precautions to prevent RNase contamination of

the reagents and degradation of the RNA sample. Find information on how to handle RNA

in the tips section starting on page 52. The tips section also provides simple guidelines

for good laboratory practice to ensure optimal performance of PCR experiments.

Important note


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Note: Blood serum and plasma are particular sample types that require special RNA purification

procedures and the amount of RNA present in the samples can usually not be accurately

determined. Due to the low levels of microRNAs and potentially high levels of inhibitors

in samples derived from serum and plasma, specific recommendations for how to set up

experiments using these types of sample can be found in the miRCURY LNA Universal RT

microRNA PCR, Instruction Manual Biofluid samples.

Normalization when running individual assays or when configuring a Pick-&-Mix microRNA PCR

panel it is important to consider how the data will be normalized. For tips and recommendationson choosing the correct reference genes and how to test and validate reference genes, please

see section on reference assays (page13) and the Tip 11 (page 58).

Excess volumes required for pipetting Liquid handling with pipettes or pipetting robots require

excess volumes of reagents due to loss during pipetting. The loss depends on the available

pipetting system but losses in the range from 10% -25% are not uncommon. All protocols in the

current instruction manual reflect the required reaction volumes and pipetting volumes should

be adjusted according to accommodate the pipetting loss of the available pipetting system.

ROX ROX is a passive reference dye used by some PCR cycles to obtain a robust read over

the entire array of wells in a 96- or 384-well PCR plate. The requirement for ROX is instrument

dependent and we recommend to follow the instrument manufactures guidelines on this (see

also Tip 8).

ABI instruments the default settings on ABI real-time PCR cyclers are not suitable for

running miRCURY LNA Universal RT microRNA PCR. Settings need to be changed from

automatic to manual background and threshold settings to obtain valid PCR data (see also Tip

10). Furthermore, if the dataset is to be analyzed using the GenEx software, it is important that

the experiment is set up as an AQ experiment, not RQ. To ensure correct settings, download

the instrument settings file at www.exiqon.com/sds.

See Tip 8

See Tip 10

See Tip 11


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RNA spike-ins consider how the RNA spike-ins should be applied in the planned study.

Please consult the section on RNA spike-ins on page 15.

Protocols for the first-strand cDNA synthesis and real-time amplication follows on the

next pages:

A. Individual assays, please go to page 20

B. Human and Mouse&Rat microRNA PCR Panels, please go to page 29

C. Focus microRNA PCR Panels, please go to page 36

D. Pick-&-Mix microRNA PCR Panels, please go to page 43

Figure 4 gives an overview and helps identifying which protocol to follow as well as the

recommended cDNA reaction volume needed for a given sample and assay type.

Figure 4. Protocol overview

Cells or tissue Serum plasma or other biofluidsSample type

Panels orprimersets:

UniversalcDNAreactions perkit

UniversalcDNAreactionvolume

miRNomepanel I

miRNomepanel I+II

1-96 97-192

Individualassays(


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Protocol A - Individual assays

This protocol is used for conducting the first-strand cDNA synthesis and real-time PCR, using

the individual assays for human, mouse and rat (product numbers 204000-205xxx).

If working with serum plasma samples or other biofluids, please refer to the specific miRCURY

LNA Universal RT microRNA PCR Instruction Manual for biofluid samples at

www.exiqon.com/serum-plasma-pcr-manual.

Before using the LNA PCR primer mix or the Reference gene primer mix for the first time,

the primers must be re-suspended: Re-suspend the primer mix by adding 220 L nuclease free water to the tube. Mix by

vortexing and spin down. Leave on ice for 20-30 minutes.

The UniSp6 RNA spike-in control primer mix is supplied with the master mix and is already

dissolved. Ensure primer concentration is 10x before proceeding with the protocol (see

page 5 for details).

Additional required materials:

96- or 384-well plate real-time PCR cycler Thermocycler for first-strand cDNA synthesis

96/384-well plates or tube strips compatible with available real-time PCR cycler

Micro centrifuge

Swing bucket centrifuge for 96-/384-well plates

Protocol-

Individualassays


21/6621


Checklist:

Have you considered excess volumes required for using liquid handling robotics see page 18

Did you consider how to use the RNA spike-ins please see page 15

ROX: The ExiLENT SYBR Green master mix, does not include the ROX passive reference

dye. Please follow instrument manufactures recommendations

ABI instruments: The use of manual background and threshold settings is necessary for

obtaining correct PCR data. Make sure to have the optimal settings by downloading the

instrument settings file at www.exiqon.com/sds. Furthermore, if the data is to be analyzed

using GenEx, the experiment must be set up as an AQ experiment, not RQ Have you optimized the input amount to the RT reaction in order to avoid inhibition?

Protocol-

In

dividualassays


22/6622


Phase I:Prepare RNA sampleSee page 52 for recommendations

Phase II: cDNA synthesisSee protocol page 23.

Phase III: real-time PCR amplificationSee protocol page 25.- Prepare adequate amount of pre-mixed primer+ PCR Master mix and distribute into wells

- Add cDNA to all primer sets to be analyzed

Phase IV: Data analysisSee data analysis guide online- Export data for further analysis- Data pre-processing, normalization

and statistical analysis

4

3

2

1

0

-1Normal Tumor Tumor

stroma

Tumor Total

miR-21

let-7a

Relativeexpression

(log2)

LNATM

primer sets

Workflow for individual primer sets (per sample)

Protocol-

Individualassays


23/6623


Protocol

The miRCURY LNA Universal RT microRNA PCR protocol is a two-part protocol consisting of:

1. First-strand cDNA synthesis (Step 1-5)

2. Real-time PCR amplification (Step 6-11)

Important: Keep reagents and reactions on ice (or at 4C) at all times.

First strand synthesis

Step 1

Dilute template RNA

Adjust each of the template RNA samples to a concentration of 5 ng/L

using nuclease free water.

Step 2

Prepare reagents

Gently thaw the 5x Reaction buffer and nuclease-free water, and

immediately place on ice. Mix by vortexing. Re-suspend the RNA spike-

ins according to the appropriate RNA Spike-ins protocol (see page 15),

leave on ice for 15-20 minutes. Immediately before use, remove the

Enzyme mix from the freezer, mix by flicking the tubes and place on

ice. Spin down all reagents.

Protocol-

In

dividualassays


24/6624


Step 4

Mix and spin

reagents

Mix the reaction by very gentle vortexing or pipetting to ensure that all

reagents are thoroughly mixed. After mixing, spin down.

Step 5

Incubate and heat

inactivate1)

Incubate for 60 min at 42C.

Heat-inactivate the reverse transcriptase

for 5 min at 95C. Immediately cool to 4C.

Store at 4C or freeze.

Step 3

Combine reagents

according to Table 2

Note: remember to

calculate necessary

excess volume for

pipetting and robotic

dead volume.

If performing first-strand cDNA synthesis on multiple RNA samples, it

is recommended to prepare an RT working solution of the 5x Reaction

buffer, water, Enzyme mix and RNA spike ins (in the proportion indicated

in the first four lines of Table 2).

The following procedure is recommended:

1. Prepare the required amount of RT working solution and place it on ice.

2. Dispense RT working solution into nuclease free tubes.

3. Dispense template RNA in each tube.

Table 2 Reverse transcription reaction setup

Reagent Volume (L),RT reaction

5x Reaction buffer 2

Nuclease-free water 4.5

Enzyme mix 1

Synthetic RNA spike ins, optionalreplace with H

2O if omitted

0.5

Template total RNA (5 ng/L) 2

Total volume 10

1) The protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20C for up to 5 weeks (optional store

at 4C for up to 4 days). It is recommended that synthesized cDNA is stored in low-nucleic acid binding tubes or plates.

Protocol-

Individualassays


25/6625


qPCR protocol

Step 6

Prepare reagents for

real-time PCR

Place cDNA (from Step 5), nuclease free water and PCR Master mix on

ice and thaw for 15-20 min. Protect the PCR Master mix vials from light.

Immediately before use, mix the PCR Master mix by pipetting up and

down. The rest of the reagents are mixed by vortexing and spun down.

Step 7

Dilute cDNA template

80x in nuclease

free water2)

Immediately before use, dilute only the amount of cDNA template

needed for the planned real-time PCR reactions 80x in nuclease free

water (e.g. add 395 L nuclease free water to each 5 L of reaction). It

is important that low-nucleic acid binding tubes or plates are used. It

is not recommended to store the 1:80 dilution of cDNA.

Recommendation: Include a passive reference dye in the cDNA

dilution if advised by instrument manufacturer. Please note that

the PCR Master mix does not include ROX. The amount of ROX

required is instrument dependent and it is important to refer to the

manufacturers recommendations when deciding how much ROX

to use, see Tip 8.

2) Adjust volumes to accommodate your in-house liquid handling system volume loss when pipetting

See Tip 8

Protocol-

In

dividualassays


26/6626


Step 9

Mix and spin

reagents

Mix the reaction by gentle pipetting to ensure that all reagents are mixed

thoroughly. After mixing cap tubes or strips or seal the plate with optical

sealing as recommended by the manufacturer. Spin down in a centrifuge

(1500g for 1 minute). The experiment can be paused at this point. Store

the reactions protected from light at 4C for up to 24 hours.

Step 8

Combine PCR Master

mix, PCR primer mix

and cDNA according

to Table 3.

Note: remember to

calculate necessary

excess volume for


dead volume.

When multiple real-time PCR reactions are performed with the same

microRNA primer set, it is recommended to prepare a primer master

mix working-solution of the PCR primers and the PCR Master mix (in

the proportion indicated in Table 3).


1. Prepare the required amount of primer:master mix working-solution

(see Table 3) and place it on ice. It is recommended to include excess

of all reagents in the master mix to compensate for pipetting excess

material.

2. Place the relevant volume of primer:master mix working-solution

in PCR tubes/wells (see Table 3) and spin tubes/plate briefly in a

centrifuge (1500g for 1 minute), to remove air bubbles.

3. Add cDNA template to each tube/well.

Table 3 Real-time PCR reaction, pr. 10 L reaction3)

Reagent Volume (L), 96/384-well plate,tubes or strips

PCR Master mix 5

PCR primer mix4) 1

Diluted cDNA template 4Total volume 10

3) If using a 96-well cycler with a minimum recommended volume of 20 L (as some ABI instruments), then use 10 L

reaction volume and set the instrument settings at 20 L.4) The PCR primer mix must be dissolved prior to real-time PCR set-up, see page 20.

Protocol-

Individualassays


27/6627


Step 10

Real-time PCR

amplification

Perform real-time PCR amplification followed by melting curve analysis

according to Table 4.

Table 4 Real-time PCR cycle conditions

Process step Settings, LC480instrument5)

Settings, otherinstruments3)

Polymerase Acti vation/Denaturation 95C, 10 min 95C, 10 min

Amplification 45 amplificationcycles at95C, 10 s60C, 1 min,ramp-rate1.6C/s6)

Optical read

40 amplificationcycles at95C, 10 s60C, 1 min,ramp-rate1.6C/s6)

Optical read

Melting curve analysis7) Yes Yes

Step 11

Analyze data

Perform initial data analysis using the software supplied with the real-

time PCR instrument to obtain raw Cq values (Cp or Ct, depending on

PCR instrument). If you are using an ABI instrument, please note that

it is not recommended to use auto Ct settings. Furthermore, if the data

is to be analyzed using Exiqon GenEx, the experiment must be set up as

an AQ experiment, not RQ. For a guide on how to set manual baseline

and threshold, refer to Tip 10, page 56 in the tips section. If you are

using a Roche LC480 instrument, we recommend analysis using the

2nd derivative method.

For tips on normalization, please see Tip 11, page 58. We

recommend performing normalization and further data analysis

with the Exiqon GenEx qPCR analysis software (www.exiqon.

com/mirna-pcr-analysis). Please refer to our data analysis guide

for recommendations.

ABI instrument user?

Apply manual baseline and threshold settings! Go to www.exiqon.com/sds to download settings file

See Tip 11

Protocol-

In

dividualassays


28/6628


5) Five additional amplification cycles are required when using the LC480 instrument to allow collection of assay datawith Cp-values up to 40.

6) The ramp-rate of cooling from 95C to 60C should be set to 1.6C/s. This is equivalent to 100% under standard cycling

conditions on the ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be

compromised.7) Melting curve analysis of the PCR product(s) is recommended to verify specificity and identity of the amplification reaction.

Melting curve analysis is an analysis step built into the software of instruments. Please follow the instructions provided by

the supplier. Note: The Tm of a PCR product depends on buf fer composition, salt concentration and the PCR instrument.

Protocol-

Individualassays


29/6629


Protocol B - Human and Mouse&RatmicroRNA PCR Panels

This protocol is used for conducting the first-strand cDNA synthesis and real-time PCR using

the following products:

Human miRNome PCR Panel (product numbers 203611 to 203614)

Mouse&Rat miRNome PCR Panel (product numbers 203709 to 203712)

If working with serum plasma samples or other biofluids, please refer to the specific

miRCURY LNA Universal RT microRNA PCR Instruction Manual for biofluid samples at


Additional required materials:

384-well plate real-time PCR cycler

Thermocycler for first-strand cDNA synthesis

Micro centrifuge

Tube for mixing water and master mix (10 ml)


Swing bucket centrifuge for 96/384-well plates

Recommended: Liquid handling robot for pipetting

Checklist:

Have you considered excess volumes required for using liquid handling

robotics see page 18


ROX: The ExiLENT SYBR Green master mix, does not include the ROX passive reference

dye. Please follow instrument manufactures recommendations

ABI instruments: The use of manual background and threshold settings is necessary

for obtaining correct PCR data. Make sure to have the optimal settings by downloading

the instrument settings file at www.exiqon.com/sds. Furthermore, if the data is to be

analyzed using GenEx, the experiment must be set up as an AQ experiment, not RQ.

Have you optimized the input amount to the RT reaction in order to avoid inhibition?

Protocol-Human,

Mo

use&RatPanels


30/6630


Workflow for Human and Mouse&Rat microRNA PCR Panels (per sample)

Protocol-Hum

an,

Mouse&RatPanels



Phase III: real-time PCR amplificationSee protocol page 33.- Mix cDNAs with PCR Master mix- Add cDNA:PCR Master mix to

PCR plates



4

3

2

1

0


stroma

TumorT otal

miR-21let-7a

Relativeexpression

(log2)


31/6631


P r

ot oc

ol

-H

um

an,

M o

use&

R a

tP

an

el s

Protocol

The miRCURY LNA Universal RT microRNA PCR protocol is a two-part protocol consisting of:




First strand synthesis:

Step 1

Dilute template RNA

Adjust each of the template RNA samples to a concentration of 5 ng/l


Step 2

Prepare reagents



in(s) according to the appropriate RNA Spike-in protocol (see page 15),





32/6632


Protocol-Hum

an,

Mouse&RatPanels

Step 4

Mix and spin

reagents



Step 5

Incubate and heat

inactivate1)



for 5 min at 95C.

Immediately cool to 4C.


Step 3

Combine reagents


Note: remember to

calculate necessary

excess volume for


dead volume.

If performing first-strand cDNA synthesis on multiple RNA samples, it

is recommended to prepare an RT working solution of the 5x Reaction

buffer, water, Enzyme mix and RNA spike ins (in the proportions indicated









Reagent Panel IVolume (L)

Panel I+IIVolume (L)

5x Reaction buffer 4 8

Nuclease-free water 9 18

Enzyme mix 2 4

Synthetic RNA spike ins, optionalreplace with H2O if omitted

1 2

Template total RNA (5ng/L) 4 8

Total volume 20 40


33/6633


Protocol-Human,

Mo

use&RatPanels

qPCR protocol:

Step 7

Combine PCR Master

mix, water and cDNA

and add to PCR plates2)

The following procedure is recommended to avoid low concentrations

of cDNA from adhering to tube surface:

1. Before removing the plate seal, briefly spin

down the plate(s) in a plate centrifuge.

2. Combine 2x PCR Master mix and water. Panel I: 2000 L 2x master

mix and 1980 L water, Panel I+II: 4000 L 2x master mix and 3960

L water.

3. Mix gently and spin down.

4. Add 20 L cDNA (panel I) or 40 L cDNA (panel I+II) and mix.

5. Add 10 L PCR Master mix: cDNA

mix to each well3).

6. Seal the plate with optical sealing as recommended by theinstrument manufacturer.

7. Spin plate briefly in a plate centrifuge (1500g for

1 minute), to to collect the sample.

The experiment can be paused at this point. Store the reactions protected

from light at 4C for up to 24 hours.

Recommendation: Include a passive reference dye in the cDNA dilution if

advised by instrument manufacturer. Please note that the PCR Master mix

does not include ROX. The amount of ROX required is instrument dependent

and it is important to refer to the manufacturers recommendations

when deciding how much ROX to use, see Tip 8.

Step 6

Prepare reagents for

real-time PCR





See Tip 8


34/6634


Step 8

Real-time PCR

amplification




Step 9

Analyze data




it is not recommended to use auto Ct settings. For a guide on how to

set manual baseline and threshold, refer to Tip 10, page 56 in the tips

section. Furthermore, if the data is to be analyzed using Exiqon GenEx,

the experiment must be set up as an AQ experiment, not RQ. Alternatively,

use ABI settings files available from www.exiqon.com/sds. If you are



For tips on normalization, please see Tip 11, page 58. We recommend

performing normalization and further data analysis with the Exiqon

GenEx qPCR analysis software (ww w.exiqon.com/mirna-pcr-analysis).

Please refer to our data analysis guide for recommendations.

Protocol-Hum

an,

Mouse&RatPanels




Settings, otherinstruments

Polymerase Activation /Denaturation 95C, 10 min 95C, 10 min


Optical read


Optical read

Melting curve analysis6) Yes Yes

See Tip 11


35/6635


Protocol-Human,

Mo

use&RatPanels

2) Adjust volumes to accommodate your in-house liquid handling system and the inaccuracy these have when pipetting.3) Corresponding to 0.05ng total RNA starting material pr. PCR reaction.4) Five additional amplification cycles are required when using the LC480 instrument to allow collection of assay data

with Cp-values up to 40.5) The ramp-rate of cooling from 95C to 60C should be set to 1.6C/s. This is equivalent to 100% under standard cycling


compromised.6) Melting curve analysis of the PCR product(s) is recommended to verify specificit y and identity of the amplification

reaction. Melting curve analysis is an analysis step built into the software of instruments. Please follow the

instructions provided by the supplier. Note: The Tm of a PCR product depends on buf fer composition, salt

concentration and the PCR instrument.


36/6636


Protocol C - Focus microRNAPCR Panels


the Cancer Focus microRNA PCR panels, 96-well plates (product numbers 203832 to 203835)

and 384-well plates (product numbers 203840 and 203841).

If working with serum plasma samples or other biofluids, please refer to the specific



Additional required materials: 96- or 384-well plate real-time PCR cycler


Micro centrifuge



Swing bucket centrifuge for 96/384-well plates


Checklist: Have you considered excess volumes required for using liquid handling robotics see page 18


ROX: The ExiLENT SYBR Green master mix does not include the ROX passive reference dye.

Please follow instrument manufactures recommendations


obtaining correct PCR data. Make sure to have the optimal settings by downloading the

instrument settings file at www.exiqon.com/sds. Furthermore, if the data is to be analyzed

using GenEx, the experiment must be set up as an AQ experiment, not RQ


Protocol-

FocusPanel

s


37/6637


Protocol-

FocusPanels



Phase III: real-time PCR amplificationSee protocol page 40.- Mix cDNAs with PCR Master mix- Add cDNA:PCR Master mix to

PCR plates



4

3

2

1

0


stroma

TumorTotal

miR-21let-7a

Relativeexpression

(log2)

Workflow for Focus microRNA PCR Panels (per sample)


38/6638


Protocol

The miRCURY LNA Universal RT microRNA PCR protocol is a two-part

protocol consisting of:





Step 1

Dilute template RNA



Step 2

Prepare reagents







Protocol-

FocusPanel

s


39/6639


Protocol-

FocusPanels

Step 3

Combine reagents


Note: remember to

calculate necessary

excess volume for


dead volume.

When performing first-strand cDNA synthesis on multiple RNA samples,

it is recommended to prepare an RT working solution of the 5x Reaction








Step 4

Mix and spin

reagents



Step 5

Incubate and

heat inactivate1)



for 5 min at 95C.





Reagent Per panel Volume (L)

5x Reaction buffer 2

Nuclease-free water 4.5

Enzyme mix 1

Synthetic RNA spike ins, optional

replace with H2O if omitted

0.5

Template total RNA (5ng/L) 2

Total volume 10


40/6640


Step 7

Combine PCR Master

mix, water and cDNA

and add to PCR plates2)

The following procedure is recommended to avoid low concentrations

of cDNA from adhering to tube surface:

1. Before removing the plate seal, briefly spin down the plate(s) in a

plate centrifuge.

2. Combine 2x PCR Master mix and water: 1000 L 2x master mix and

990 L water

(Focus panel consisting of 2x 96 assays)

3. Mix gently and spin down.

4. Add 10 L cDNA (Focus panel consisting of 2x 96 assays).

5. Add 10 L PCR Master mix: cDNA mix to each well3).

6. Seal the plate with optical sealing as recommended by the

instrument manufacturer.








Step 6

Prepare reagents

for real-time PCR





qPCR protocol:

2) Adjust volumes to accommodate your in-house liquid handling system and the losses these have when pipetting.3) Corresponding to 0.05ng total RNA starting material pr. PCR reaction.

Protocol-

FocusPanel

sSee Tip 8


41/6641


Step 8

Spin plate

Spin plate briefly in a plate centrifuge (1500g for 1 minute), to collect

the sample.

Step 9

Real-time PCR

amplification





Settings, otherinstruments4)

PolymeraseActivation/Denaturation

95C, 10 min 95C, 10 min


Optical read


Optical read

Melting curveanalysis7)

Yes Yes

4) If using a 96-well cycler with a minimum recommended volume of 20 L (like some ABI instruments), then use 10 L

reaction volume and set the instrument settings at 20 L.5) Five additional amplification cycles is required when using the LC480 instrument to allow collection of assay data with

Cp-values up to 40.6) The ramp-rate of cooling from 95C to 60C should be set to 1.6C/s. This is equivalent to 100% under standard cycling


compromised.7) Melting curve analysis of the PCR product(s) is recommended to verify specificity and identity of the amplification reaction.

Melting curve analysis is an analysis step built into the software of instruments. Please follow the instructions provided by

the supplier. Note: The Tm of a PCR product depends on buf fer composition, salt concentration and the PCR instrument.

Protocol-

FocusPanels




42/6642


Step 10

Analyze data




it is not recommended to use auto Ct settings. Furthermore, if the data

is to be analyzed using Exiqon GenEx, the experiment must be set up as

an AQ experiment, not RQ. For a guide on how to set manual baseline

and threshold, refer to Tip 10, page 56 in the tips section. If you are



For tips on normalization, please see Tip 11, page 58. We recommend

performing normalization and further data analysis with the Exiqon

GenEx qPCR analysis software (ww w.exiqon.com/mirna-pcr-analysis).

Please refer to our data analysis guide for recommendations.

Protocol-

FocusPanel

s

See Tip 11


43/6643


Protocol-

Pic

k-&-MixPanels

Protocol D - Pick-&-Mix microRNAPCR Panels


the Pick-&-Mix microRNA PCR Panel, 96-well plates (product number 203801) and 384-well

plates (product number 203802).

If working with serum plasma samples or biofluid samples, please refer to the specific



Additional required materials: 96- or 384-well plate real-time PCR cycler


Micro centrifuge


Swing bucket centrifuge for 96/384-well plates.



Checklist: Have you considered excess volumes required for using liquid handling robotics

see page 18


ROX: The ExiLENT SYBR Green master mix does not include the ROX passive reference dye.

Please follow instrument manufactures recommendations


obtaining correct PCR data. Furthermore, if the data is to be analyzed using GenEx, the

experiment must be set up as an AQ experiment, not RQ. Make sure to have the optimal

settings by downloading the instrument settings file at www.exiqon.com/sds.



44/6644




Phase III: real-time PCR amplificationSee protocol page 48.- Mix cDNAs with PCR Master mix- Add cDNA:PCR Master mix to primer

sets replicates (indicated by colored boxes)



4

3

2

1

0


stroma

TumorT otal

miR-21let-7a

Relativeexpression

(log2)

Workflow for Pick-&-Mix PCR plates (per sample)

Protocol-

Pick-&-MixPanels


45/6645


Pick-&-Mix microRNA PCR Panel layouts

Overview of the pre-defined plate layouts

available for 96-well and 384-well plates of

the Pick-&-Mix Panel. Wells in dark gray and

light greay are pre-occupied by interplate-

calibrators (UniSp3 IPC) and RNA spike-in

controls (UniSp6), respectively.

10 microRNAs x 8 samples

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 CP IPC

1 2 3 4 5 6 7 8 9 10 CP IPC

1 2 3 4 5 6 7 8 9 10 CP IPC

1 2 3 4 5 6 7 8 9 10 CP IPC

1 2 3 4 5 6 7 8 9 10 CP IPC

1 2 3 4 5 6 7 8 9 10 CP IPC

1 2 3 4 5 6 7 8 9 10 CP IPC

1 2 3 4 5 6 7 8 9 10 CP IPC


A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 11 IPC

13 14 15 16 17 18 19 20 21 22 23

13 14 15 16 17 18 19 20 21 22 23

13 14 15 16 17 18 19 20 21 22 23

13 14 15 16 17 18 19 20 21 22 23

CP

1 2 3 4 5 6 7 8 9 10 11 IPC

CP

1 2 3 4 5 6 7 8 9 10 11 IPC

CP

1 2 3 4 5 6 7 8 9 10 11 IPC

CP

92 microRNAs x 1 sample

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 11 IPC

13 14 15 16 17 18 19 20 21 22 23

25 26 27 28 29 30 31 32 33 34 35

37 38 39 40 41 42 43 44 45 46 47

49 50 51 52 53 54 55 56 57 58 59

61 62 63 64 65 66 67 68 69 70 71

73 74 75 76 77 78 79 80 81 82 83

85 86 87 88 89 90 91 92 93 94 95

CP

IPC

IPC

60

72

84

96


A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 241 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 15 1 6 1 7 1 8 19 2 0 21 2 2 C P IPC

C P I PC

C P I PC

C P I PC

C P I PC

C P I PC

C P I PC

C P I PC

C P I PC

C P I PC

C P I PC

C P I PC

C P I PC

C P I PC

C P I PC

C P I PC

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 21 2 2

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 21 2 2

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 21 2 2

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 21 2 2

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 21 2 2

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2


A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

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

1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 19 20 21 22 23 CP

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 19 2 0 21 2 2 2 3

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 19 2 0 21 2 2 2 3

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 19 2 0 21 2 2 23

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 19 2 0 21 2 2 23

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 19 2 0 21 2 2 23

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 1 9 2 0 21 2 2 23

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 1 9 2 0 21 2 2 23

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

IPC

CP

IPC

CP

IPC

CP

IPC

CP

IPC

CP

IPC

CP

IPC

CP

IPC


A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 241 1 22 33 44 55 66 77 88 99 1010 1111 1212

1 1 22 33 44 55 66 77 88 99 1010 1111 1212

13 13 1414 1515 1616 1717 1818 1919 2020 2121 2222 2323 2424

13 13 1414 1515 1616 1717 1818 1919 2020 2121 2222 2323 2424

25 25 2626 2727 2828 2929 3030 3131 3232 3333 3434 CPCP 3636

25 25 2626 2727 2828 2929 3030 3131 3232 3333 3434 CPCP 3636

37 37 3838 3939 4040 4141 4242 4343 4444 4545 4646 4747 4848

37 37 3838 3939 4040 4141 4242 4343 4444 4545 4646 4747 4848

49 49 5050 5151 5252 5353 5454 5555 5656 5757 5858 5959 6060

49 49 5050 5151 5252 5353 5454 5555 5656 5757 5858 5959 6060

61 61 6262 6363 6464 6565 6666 6767 6868 6969 7070 IPCIPC 7272

61 61 6262 6363 6464 6565 6766 6767 6868 6969 7070 IPCIPC 7272

73 73 7474 7575 7676 7777 7878 7979 8080 8181 8282 8383 8484

73 73 7474 7575 7676 7777 7878 7979 8080 8181 8282 8383 8484

85 85 8686 8787 8888 8989 9090 9191 9292 9393 9494 9595 9696

85 85 8686 8787 8888 8989 9090 9191 9292 9393 9494 9595 9696

380 microRNAs x 1 sample

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 241 2 43 65 87 109 1211 1413 1615 1817 2019 2221 2423

25 26 2827 3029 3231 3433 3635 3837 4039 4241 4443 4645 4847

5049 51 5352 5554 5756 5958 6160 6362 6564 6766 6968 7170 72

73 74 7675 7877 8079 8281 8483 8685 8887 9089 9291 9493 IPC95

97 98 10099 102101 104103 106105 108107 110109 112111 114113 116115 118117 120119

121 122 124123 126125 128127 130129 132131 134133 136135 138137 140139 142141 144143

145 146 148147 150149 152151 154153 156155 158157 160159 162161 164163 166165 168167

169 170 172171 174173 176175 178177 180179 182181 184183 186185 188187 190189 IPC191

193 194 196195 198197 200199 202201 204203 206205 208207 210209 212211 214213 216215

217 218 220219 222221 224223 226225 228227 230229 232231 234233 236235 238237 240239

241 242 244243 246245 248247 250249 252251 254253 256255 258257 260259 262261 264263

265 266 268267 270269 272271 274273 276275 278277 280279 282281 284283 286285 CP287

289 290 292291 294293 296295 298297 300299 302301 304303 306305 308307 310309 312311

313 314 316315 318317 320319 322321 324323 326325 328327 330329 332331 334333 336335

337 338 340339 342341 344343 346345 348347 350349 352351 354353 356355 358357 360359

361 362 364363 366365 368367 370369 372371 374373 376375 378377 380379 382381 IPC383

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Protocol

The miRCURY LNA Universal RT microRNA PCR protocol is a two-part

protocol consisting of:





Step 1

Dilute template RNA



Step 2

Prepare reagents







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Step 3

Combine reagents


When performing first-strand cDNA synthesis on multiple RNA samples,

it is recommended to prepare an RT working solution of the 5x Reaction







Table 9 Reverse transcription reaction setup per sample1)

The amount of 100x fold diluted cDNA needed for the different

Pick-&-Mix layouts can be seen in Table 10 Step 7.

Reagent Volume (L)< 100 miRNAanalyzed persample

Volume (L)> 100 miRNAanalyzed persample

5x Reaction buffer 2 4

Nuclease-free water 4.5 9

Enzyme mix 1 2

Synthetic RNA spike ins, optionalreplace with H

2O if omitted

0.5 1

Template total RNA(5ng/L)

2 4

Total volume 10 20

Step 4

Mix and spin reagents



1) The amount of cDNA required per sample depends on the number of microRNAs analyzed per sample. The volumes

suggested here provides excess amount of cDNA, which ensures the highest possible reproducibility because these

volumes can be pipetted with great accuracy.

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2) Although not recommended, the protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20C for

up to 5 weeks (optional store at 4C for up to 4 days). It is recommended that synthesized cDNA be stored in low-nucleic

acid binding tubes or plates.

Step 5

Incubate and

heat inactivate2)



for 5 min at 95C.



qPCR protocol:

Step 6

Prepare reagents

for real-time PCR

Place cDNA (from Step 5), nuclease free water and PCR Master

mix on ice and thaw for 15-20 min. Protect the PCR Master mix

vials from light. Immediately before use, mix the PCR Master mix

by pipetting up and down. The rest of the reagents are mixed by vortexing

and spun down.

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Step 7

Dilute cDNA template

100x in nuclease

free water3)

Dilute the cDNA from the RT reactions to give a final 100x dilution.

Suggested cDNA dilution procedures is shown in Table 10 along with

required volumes of diluted cDNA for the different Pick-&-Mix pre-

defined layouts. It is receommeded that low-nucleic acid binding.

For fully customized layouts dilutions may be adjusted to the specific

replicate scheme. tubes or plates are used. It is not recommended to

store the 1:100 dilution of cDNA.

Table 10 Amount of 100x diluted cDNA needed in Pick-&-Mix plates

Numbers in parenthesis designate total number of assays/sample,

including controls and inter-plate calibrators. Loss from pipetting is not

included in the volumes. listed in the right column (diluted cDNA needed).






Pick-&-Mix plateconfiguration

Suggested cDNAdilution procedurecDNA + nucleasefree water (L)

Volume (L) of dilutedcDNA needed for eachPick-&-Mix plate

8x10 (12) 2 + 198 60

4x22 (24) 2 + 198 120

1x92 (96) 5 + 495 480

16x22 (24) 2 + 198 120

8x46 (48) 3 + 297 240

4x94 (96) 5 + 495 4801x380 (384) 20 + 1980 1920

3) Adjust volumes to accommodate your in-house liquid handling system and the losses these have when pipetting.

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Step 8

Combine cDNA and

PCR Master mix 1:1

and add to PCR plates


1. Before removing the plate seal, briefly spin down the plate(s) in a

plate centrifuge.

2. Combine 2x PCR Master mix and 100x diluted cDNA 1:1 (e.g. 500 L

2x PCR master mix and 500 L diluted cDNA).

3. Mix gently by inverting the tube, spin down.

4. Add 10 L PCR Master mix:

cDNA mix to each well.4)

5. Seal the plate with optical sealing as recommended by the

instrument manufacturer.



Step 9Spin plate

Spin plate briefly in a plate centrifuge (1500g for 1 minute), to removeair bubbles.

Step 10

Real-time

PCR amplification




Process step Settings, LC480

instrument

5)

Settings, other

instruments

6)

Polymerase Acti vation/Denaturation 95C, 10 min 95C, 10 min


Optical read


Optical read

Melting curveanalysis8)

Yes Yes

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Step 11

Analyze data




it is not recommended to use auto Ct settings. For a guide on how to

set manual baseline and threshold, refer to Tip 10, page 56 in the tips

section. Furthermore, if the data is to be analyzed using Exiqon GenEx,

the experiment must be set up as an AQ experiment, not RQ.

For tips on normalization, please see Tip 11, page 58. If you are using

a Roche LC480 instrument, we recommend analysis using the 2nd

derivative method. We recommend performing normalization and

further data analysis with the Exiqon GenEx qPCR analysis software

(ww w.exiqon.com/mirna-pcr-analysis). Please refer to our data analysis

guide for recommendations.

4) Corresponding to 0.05ng total RNA starting material pr. PCR reaction.5)Five additional amplification cycles are required when using the LC480 instrument to allow collection of assay data with

Cp-values up to 40.6)If using a 96-well cycler with a minimum recommended volume of 20 L (like some ABI instruments), then use 10

Documents

MiRCURY LNA PCR System Manual