TaqMan MicroRNA Assays Protocol

DRAFT

December 23, 2005 11:22 am, miRNA_Title.fm

TaqMan

MicroRNA Assays

Protocol

TaqMan

MicroRNA Assays

Protocol

DRAFT

December 23, 2005 11:22 am, miRNA_Title.fm

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

Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that

may appear in this document. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential

damages in connection with or arising from the use of this document.

NOTICE TO PURCHASER: LIMITED LICENSE

A license to perform the 5' nuclease process for research requires the use of a Licensed 5' Nuclease Kit (containing Licensed

Probe), or the combination of an Authorized 5' Nuclease Core Kit plus Licensed Probe, or license rights that may be purchased

from Applied Biosystems. This product contains Licensed Probe. Its purchase price includes a limited, non-transferable immunity

from suit under U.S. Patents Nos. 5,538,848, 5,723,591, 5,876,930, 6,030,787, and 6,258,569, and corresponding patent claims

outside the United States, owned by the Applera Corporation, and U.S. Patent No. 5,804,375 (claims 1-12 only) and corresponding

patent claims outside the United States, owned by Roche Molecular Systems, Inc. or F. Hoffmann-La Roche Ltd (ìRocheî), for

using only this amount of probe in the practice of the 5í nuclease process solely for the purchaserís own internal research. This

product is also a Licensed Probe for use with service sublicenses available from Applied Biosystems. This product conveys no

rights under U.S. Patents Nos. 5,210,015 and 5,487,972, or corresponding patent claims outside the United States, which claim 5í

nuclease processes, or under U.S. Patents Nos. 5,476,774 and 5,219,727, or corresponding patent claims outside the United States,

which claim quantification methodology, expressly, by implication, or by estoppel. No right under any other patent claims (such as

apparatus or system claims) and no right to perform commercial services of any kind, including without limitation reporting the

results of purchaser's activities for a fee or other commercial consideration, is hereby granted expressly, by implication, or by

estoppel. This product is for research use only. Diagnostic uses require a separate license from Roche.

Notice to Purchaser: Disclaimer of License

The TaqManÆ microRNA Assay is optimized for use in the polymerase chain reaction (PCR) covered by patents owned by Roche

Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. No license under these patents to use the PCR Process is conveyed

expressly or by implication to the purchaser by the purchase of these products. A license to use the PCR Process for the purchaser's

own internal research accompanies the purchase of certain Applied Biosystems reagents when used in conjunction with an

authorized thermal cycler, or is available from Applied Biosystems.

Trademarks

AB (Design), ABI P

RISM

, Applied Biosystems, and AmpErase are registered trademarks and Applera, FAM, ROX, MicroAmp, and

MultiScribe are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries.

AmpliTaq Gold and TaqMan are registered trademarks of Roche Molecular Systems, Inc.

All other trademarks are the property of their respective owners.

Part Number 4364031 Rev. B

01/2006

DRAFT

December 23, 2005 12:24 pm, 4364031bTOC.fm

Contents

TaqMan MicroRNA Assays Protocol iii

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Section 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

About MicroRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

About this Product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Available TaqMan MicroRNA Assays . . . . . . . . . . . . . . . . . . . . . . . . . 2

About This Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Chemistry Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Two-Step RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

About the Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

5´ Nuclease Assay Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Materials and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Available Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Materials and Equipment Not Included . . . . . . . . . . . . . . . . . . . . . . . 6

Preventing Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

General PCR Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Procedural Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Section 2 Using Individual Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

RNA Template Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Per Reaction Input Amount of Total RNA . . . . . . . . . . . . . . . . . . . . . 11

Preparing the RT Reaction Master Mix . . . . . . . . . . . . . . . . . . . . . . 12

Preparing the RT Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Performing Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . 14

DRAFT

December 23, 2005 12:24 pm, 4364031bTOC.fm

iv TaqMan MicroRNA Assays Protocol

PCR Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

PCR Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Reagent Preparation Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

PCR Reaction Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Preparing the PCR Reaction Plate . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Setting Up the Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Running the Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Section 3 Analyzing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

General Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Tools for Analyzing TaqMan MicroRNA Assay Results . . . . . . . . . . 21

Resources for Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

DRAFT

December 23, 2005 12:24 pm, miRNA_preface.fm

TaqMan MicroRNA Assays Protocol v

Preface

This preface contains:

Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ix

Safety

Safety Alert Words

Four safety alert words appear in Applied Biosystems user

documentation at points in the document where you need to be aware

of relevant hazards. Each alert word–IMPORTANT, CAUTION,

WARNING, DANGER–implies a particular level of observation or

action, as defined below:

IMPORTANT!

– Indicates information that is necessary for proper

instrument operation, accurate chemistry kit use, or safe use of a

chemical.

– Indicates a potentially hazardous situation that,

if not avoided, may result in minor or moderate injury. It may also be

used to alert against unsafe practices.

– Indicates a potentially hazardous situation that,

if not avoided, could result in death or serious injury.

– Indicates an imminently hazardous situation

that, if not avoided, will result in death or serious injury. This signal

word is to be limited to the most extreme situations.

Chemical Hazard Warning

CHEMICAL HAZARD. Some of the chemicals

used with Applied Biosystems instruments and protocols are

potentially hazardous and can cause injury, illness, or death.

DRAFT

December 23, 2005 12:24 pm, miRNA_preface.fm

vi TaqMan MicroRNA Assays Protocol

Chemical Safety Guidelines

To minimize the hazards of chemicals:

• Read and understand the Material Safety Data Sheets (MSDSs)

provided by the chemical manufacturer before you store, handle,

or work with any chemicals or hazardous materials. (See “About

MSDSs” below.)

• Minimize contact with chemicals. Wear appropriate personal

protective equipment when handling chemicals (for example,

safety glasses, gloves, or protective clothing). For additional

safety guidelines, consult the MSDS.

• Minimize the inhalation of chemicals. Do not leave chemical

containers open. Use only with adequate ventilation (for

example, fume hood). For additional safety guidelines, consult

the MSDS.

• Check regularly for chemical leaks or spills. If a leak or spill

occurs, follow the manufacturer’s cleanup procedures as

recommended on the MSDS.

• Comply with all local, state/provincial, or national laws and

regulations related to chemical storage, handling, and disposal.

About MSDSs

Chemical manufacturers supply current Material Safety Data Sheets

(MSDSs) with shipments of hazardous chemicals to new customers.

They also provide MSDSs with the first shipment of a hazardous

chemical to a customer after an MSDS has been updated. MSDSs

provide the safety information you need to store, handle, transport,

and dispose of the chemicals safely.

Each time you receive a new MSDS packaged with a hazardous

chemical, be sure to replace the appropriate MSDS in your files.

Obtaining MSDSs

The MSDS for any chemical supplied by Applied Biosystems is

available to you free 24 hours a day. To obtain MSDSs:

1. Go to https://docs.appliedbiosystems.com/msdssearch.html

2. In the Search field of the MSDS Search page:

a. Type in the chemical name, part number, or other

information that you expect to appear in the MSDS of

interest.

DRAFT

December 23, 2005 12:24 pm, miRNA_preface.fm

TaqMan MicroRNA Assays Protocol vii

b. Select the language of your choice.

c. Click Search.

3. To view, download, or print the document of interest:

a. Right-click the document title.

b. Select:

• Open – To view the document

• Save Target As – To download a PDF version of the

document to a destination that you choose

• Print Target – To print the document

4. To have a copy of an MSDS sent by fax or e-mail, in the Search

Results page:

a. Select Fax or Email below the document title.

b. Click RETRIEVE DOCUMENTS at the end of the

document list.

c. Enter the required information.

d. Click View/Deliver Selected Documents Now.

Note:

For the MSDSs of chemicals not distributed by Applied

Biosystems, contact the chemical manufacturer.

Chemical Waste Hazard

CHEMICAL WASTE HAZARD. Some wastes

produced by the operation of the instrument or system are potentially

hazardous and can cause injury, illness, or death.

Chemical Waste Safety Guidelines

To minimize the hazards of chemical waste:

• Read and understand the Material Safety Data Sheets (MSDSs)

provided by the manufacturers of the chemicals in the waste

container before you store, handle, or dispose of chemical waste.

• Provide primary and secondary waste containers. (A primary

waste container holds the immediate waste. A secondary

container contains spills or leaks from the primary container.

Both containers must be compatible with the waste material and

meet federal, state, and local requirements for container

storage.)

DRAFT

December 23, 2005 12:24 pm, miRNA_preface.fm

viii TaqMan MicroRNA Assays Protocol

• Minimize contact with chemicals. Wear appropriate personal

protective equipment when handling chemicals (for example,

safety glasses, gloves, or protective clothing). For additional

safety guidelines, consult the MSDS.

• Minimize the inhalation of chemicals. Do not leave chemical

containers open. Use only with adequate ventilation (for

example, fume hood).For additional safety guidelines, consult

the MSDS.

• Handle chemical wastes in a fume hood.

• After emptying the waste container, seal it with the cap

provided.

• Dispose of the contents of the waste tray and waste bottle in

accordance with good laboratory practices and local,

state/provincial, or national environmental and health

regulations.

Waste Disposal

If potentially hazardous waste is generated when you operate the

instrument, you must:

• Characterize (by analysis if necessary) the waste generated by

the particular applications, reagents, and substrates used in your

laboratory.

• Ensure the health and safety of all personnel in your laboratory.

• Ensure that the instrument waste is stored, transferred,

transported, and disposed of according to all local,

state/provincial, and/or national regulations.

IMPORTANT!

Radioactive or biohazardous materials may require

special handling, and disposal limitations may apply.

Biological Hazard Safety

BIOHAZARD. Biological samples such as

tissues, body fluids, infectious agents, and blood of humans and other

animals have the potential to transmit infectious diseases. Follow all

applicable local, state/provincial, and/or national regulations. Wear

appropriate protective equipment, which includes but is not limited

to: protective eyewear, face shield, clothing/lab coat, and gloves. All

work should be conducted in properly equipped facilities using the

appropriate safety equipment (for example, physical containment

DRAFT

December 23, 2005 12:24 pm, miRNA_preface.fm

TaqMan MicroRNA Assays Protocol ix

devices). Individuals should be trained according to applicable

regulatory and company/institution requirements before working

with potentially infectious materials. Read and follow the applicable

guidelines and/or regulatory requirements in the following:

• U.S. Department of Health and Human Services guidelines

published in Biosafety in Microbiological and Biomedical

Laboratories (stock no. 017-040-00547-4;

http://bmbl.od.nih.gov)

• Occupational Safety and Health Standards, Bloodborne

Pathogens (29 CFR§1910.1030; http://www.access.gpo.gov/

nara/cfr/waisidx_01/ 29cfr1910a_01.html).

• Your company’s/institution’s Biosafety Program protocols for

working with/handling potentially infectious materials.

Additional information about biohazard guidelines is available at:

http://www.cdc.gov

How to Obtain

Support

For the latest services and support information for all locations, go to

http://www.appliedbiosystems.com, then click the link for

Support.

At the Support page, you can:

• Search through frequently asked questions (FAQs)

• Submit a question directly to Technical Support

• Order Applied Biosystems user documents, MSDSs, certificates

of analysis, and other related documents

• Download PDF documents

• Obtain information about customer training

• Download software updates and patches

In addition, the Support page provides access to worldwide telephone

and fax numbers to contact Applied Biosystems Technical Support

and Sales facilities.

DRAFT

December 23, 2005 12:24 pm, miRNA_preface.fm

x TaqMan MicroRNA Assays Protocol

DRAFT

December 23, 2005 12:24 pm, Intro-Chem.fm

TaqMan MicroRNA Assays Protocol 1

Introduction

Section 1 Introduction

Overview

Purpose

The TaqMan

MicroRNA Assays are designed to detect and

accurately quantify mature microRNAs (miRNAs) using Applied

Biosystems real-time PCR instruments.

TaqMan MicroRNA Assays offer several distinct advantages over

conventional miRNA-detection methods, including:

• High-quality quantitative data – The assays can detect and

quantify miRNA over more than six logs of dynamic range.

• Sensitivity – The assays can detect miRNAs in as little as 1 to

10 ng of total RNA, allowing you to conserve limited samples.

• High Specificity – The assays detect only mature miRNA, not

its precursor, with single-base discrimination.

• Fast and simple methodology– The two-step protocol takes less

than four hours and can be used with any Applied Biosystems

Real-Time PCR instrument.

About MicroRNAs

MicroRNAs are endogenous RNAs, about 22 nucleotides long, that

play important regulatory roles in animals and plants by targeting

mRNA transcripts for cleavage or translational repression (Bartel,

2004). To date, hundreds of unique, mature miRNAs have been

identified across species, with more continuing to be discovered.

Their expression levels vary greatly among species and tissues (Kim

et al., 2004).

Low abundant miRNAs have been difficult to detect based on current

technologies, such as cloning, Northern hybridization (Lim et al.,

2003), microarrays, and other techniques.

DRAFT

December 23, 2005 12:24 pm, Intro-Chem.fm

2 TaqMan MicroRNA Assays Protocol

About this

Product

The TaqMan MicroRNA Assays use looped-primer RT-PCR, a new

real-time quantification method, to accurately detect mature

miRNAs.

Each TaqMan MicroRNA assay includes:

• One tube containing miRNA-specific RT primer

• One tube containing a mix of:

– miRNA-specific forward PCR primer

– specific reverse PCR primer

– miRNA-specific TaqMan MGB probe

Available TaqMan

MicroRNA

Assays

The TaqMan MicroRNA Assays are available for a range of species.

Because many mature miRNA sequences are identical across related

species, many assays for human are also valid for mouse and rat.

Contact technical support or visit www.appliedbiosystems.com for

details.

For the most current list of assays, refer to the Applied Biosystems

website:

http://www.appliedbiosystems.com

About This

Protocol

This protocol provides:

• Background information about the TaqMan MicroRNA Assays

• A list of equipment and materials needed for the protocol

• Procedures for using TaqMan MicroRNA assays

DRAFT

December 23, 2005 12:24 pm, Intro-Chem.fm

Chemistry Overview

TaqMan MicroRNA Assays Protocol 3

Introduction

Chemistry Overview

Two -S t ep

RT-PCR

Quantification using the TaqMan MicroRNA Assays is done using

two-step RT-PCR:

1. In the reverse transcription (RT) step, cDNA is reverse

transcribed from total RNA samples using specific miRNA

primers from the TaqMan MicroRNA Assays and reagents from

the TaqMan

MicroRNA Reverse Transcription Kit.

2. In the PCR step, PCR products are amplified from cDNA

samples using the TaqMan MicroRNA Assay together with the

TaqMan

Universal PCR Master Mix.

Figure 1 Two-step RT-PCR

3′

5′

miRNA

Forward primer

Looped

RT prime

Extension of primer on miRNA

Extension of primer on cDNA

Synthesis of second cDNA strand

PCR amplification of cDNA

Synthesis of first cDNA strand

5′3′

Cycle #2

Cycle #1

5′

3′

3′5′

Forward primer

Reverse primer

5′3′

Step 1 = RT

Step 2 = Real-Time PCR

DRAFT

December 23, 2005 12:24 pm, Intro-Chem.fm

4 TaqMan MicroRNA Assays Protocol

About the Probes

The TaqMan MGB probes contain:

• A reporter dye (FAM

™

dye) linked to the 5′ end of the probe

• A minor groove binder (MGB) at the 3´ end of the probe

This modification increases the melting temperature (T

)

without increasing probe length (Afonina et al., 1997; Kutyavin

et al., 1997), which allows the design of shorter probes.

• A nonfluorescent quencher (NFQ) at the 3′ end of the probe

Because the quencher does not fluoresce, Applied Biosystems

sequence detection systems can measure reporter dye

contributions more accurately.

5´ Nuclease

Assay Process

The 5′ nuclease assay process (Figures 2 through 6) takes place

during PCR amplification. This process occurs in every cycle and

does not interfere with the exponential accumulation of product.

Figure 2 Legend for 5′ nuclease assay process figures

During PCR, the TaqMan MGB probe anneals specifically to a

complementary sequence between the forward and reverse primer

sites (Figure 3).

When the probe is intact (Figures 3 and 4), the proximity of the

reporter dye to the quencher dye results in suppression of the reporter

fluorescence primarily by Förster-type energy transfer (Förster, 1948;

Lakowicz, 1983).

Figure 3 Polymerization

MGB

NFQ

= Nonfluorescent quencher

= Minor groove binder

= Reporter

= Hot-start DNA polymerase

5′

3′

5′

5′ 3′

5′

Forward

Primer

Reverse

Primer

MGB

NFQ

DRAFT

December 23, 2005 12:24 pm, Intro-Chem.fm

Chemistry Overview

TaqMan MicroRNA Assays Protocol 5

Introduction

Figure 4 Strand displacement

The DNA polymerase cleaves only probes that are hybridized to the

target (Figure 5). Cleavage separates the reporter dye from the

quencher dye; the separation of the reporter dye from the quencher

dye results in increased fluorescence by the reporter. The increase in

fluorescence signal occurs only if the target sequence is

complementary to the probe and is amplified during PCR. Because of

these requirements, nonspecific amplification is not detected.

Figure 5 Cleavage

Polymerization of the strand continues, but because the 3′ end of the

probe is blocked, there is no extension of the probe during PCR

(Figure 6).

Figure 6 Completion of polymerization

5′3′

5′

5′ 3′

5′

Forward

Primer

Reverse

Primer

MGB

NFQ

5′

Forward

Primer

5′

3′

5′ 3′

5′

Reverse

Primer

MGB

NFQ

3′

5′

Reverse

Primer

3′

5′

Forward

Primer

MGB

NFQ

DRAFT

December 23, 2005 12:24 pm, Intro-Chem.fm

6 TaqMan MicroRNA Assays Protocol

Materials and Equipment

Available

Products

The TaqMan MicroRNA Assays are available for a range of species.

For the most current list of assays, refer to the Applied Biosystems

website:

http://www.appliedbiosystems.com

Each assay consists of:

• One tube of RT primer

• One tube of TaqMan Assay (preformulated forward/reverse

primer and MGB probe)

• Enough material for 150 Real-Time PCR reactions (at a total

reaction volume of 20 µL)

IMPORTANT!

TaqMan MicroRNA Assays are specifically optimized

to work with the MuLV Reverse Transcriptase contained in the

TaqMan MicroRNA Reverse Transcription Kit. Applied Biosystems

cannot guarantee assay performance if you use other reverse

transcriptase enzymes.

Additionally, Applied Biosystems recommends that you use

TaqMan

2✕ Universal PCR Master Mix, No AmpErase

UNG with

TaqMan MicroRNA Assays.

Storage

Store TaqMan MicroRNA Assays at −15 to −25 °C.

Materials and

Equipment Not

Included

Table 1 includes required and optional equipment and materials for

using TaqMan MicroRNA Assays. Unless otherwise noted, many

items listed are available from major laboratory suppliers.

Table 1 User-supplied materials and equipment

Materials and Equipment Source

Required

TaqM a n

MicroRNA Reverse Transcription Kit

• 200 reactions

• 1000 reactions

Applied Biosystems

• PN 4366596

• PN 4366597

TaqM a n

2✕ Universal PCR Master Mix, No

AmpErase

UNG

Applied Biosystems

PN 4324018

DRAFT

December 23, 2005 12:24 pm, Intro-Chem.fm

Materials and Equipment

TaqMan MicroRNA Assays Protocol 7

Introduction

Additional Materials and Equipment

Applied Biosystems 7900HT Fast Real-Time PCR

System

Contact your local

Applied Biosystems

sales office.

Applied Biosystems 7300/7500/7500 Fast Real-

Time PCR System

GeneAmp

PCR System 9700 thermal cycler

ABI P

RISM

96-Well Optical Reaction Plate with

Barcode (code 128)

Applied Biosystems

PN 4326659

ABI P

RISM

384-Well Clear Optical Reaction Plate

with Barcode (code 128)

Applied Biosystems

PN 4309849

ABI P

RISM

Optical Adhesive Covers

(quantity 100)

Applied Biosystems

PN 4311971

Optical Adhesive Covers (quantity 25) Applied Biosystems

PN 4360954

ABI P

RISM

Optical Caps, 8 caps/strip Applied Biosystems

PN 4323032

ABI P

RISM

Cap Installing Tool Applied Biosystems

PN 4330015

Adhesive Seal Applicator Kit Applied Biosystems

PN 4333183

MicroAmp

™

Clear Adhesive Films Applied Biosystems

PN 4306311

Optical Compression Pads (quantity 5) Applied Biosystems

PN 4312639

Applied Biosystems Reagent Tubes With Caps,

10-mL

Applied Biosystems

PN 4305932

Centrifuge with plate holders Major Laboratory

Supplier (MLS)

Disposable gloves MLS

Microcentrifuge MLS

Pipette tips, aerosol resistant, nuclease-free: 1- to

20-µL range, 20- to 200-µL range, 100- to

1000-µL range

MLS

Pipettors (positive-displacement, air-

displacement, or multichannel): 1- to 20-µL range,

20- to 200-µL range, 100- to 1000-µL range

MLS

Polypropylene tubes MLS

Table 1 User-supplied materials and equipment (continued)

Materials and Equipment Source

DRAFT

December 23, 2005 12:24 pm, Intro-Chem.fm

8 TaqMan MicroRNA Assays Protocol

RNase-free, sterile-filtered water MLS

Vortexer MLS

Microsoft

Excel or equivalent spreadsheet and

analysis software

Software suppliers

a. TaqMan MicroRNA Assays are specifically optimized to work with the

MuLV Reverse Transcriptase contained in the TaqMan MicroRNA Reverse

Transcription Kit. Applied Biosystems cannot guarantee assay

performance if you use other reverse transcriptase enzymes.

b. Applied Biosystems strongly recommends that you use these Applied

Biosystems reagents with the TaqMan MicroRNA Assays.

Table 2 Applied Biosystems documents

Documents Part Number

TaqMan

MicroRNA Reverse Transcription Kit

Product Insert

4367038

Applied Biosystems 7900HT Fast Real-Time PCR

System and SDS Enterprise Database User Guide

4351684

Applied Biosystems 7300/7500/7500 Fast Real-

Time PCR System Installation and Maintenance

Getting Started Guide

4347828

Applied Biosystems 7300/7500/7500 Fast Real-

Time PCR System Absolute Quantification Getting

Started Guide

4347825

Real-Time PCR Systems Chemistry Guide 4348358

Table 1 User-supplied materials and equipment (continued)

Materials and Equipment Source

DRAFT

December 23, 2005 12:24 pm, Intro-Chem.fm

Preventing Contamination

TaqMan MicroRNA Assays Protocol 9

Introduction

Preventing Contamination

Overview

PCR assays require special laboratory practices to avoid false

positive amplifications (Kwok and Higuchi, 1989). The high

throughput and repetition of these assays can lead to amplification of

a single DNA molecule (Saiki et al., 1985; Mullis and Faloona,

1987).

General PCR

Practices

General PCR practices to prevent contamination:

• Maintain separate areas, dedicated equipment, and supplies for:

– Sample preparation

– PCR setup

– PCR amplification

– Analysis of PCR products

• Do not bring amplified PCR products into the PCR setup area.

• Wear a clean lab coat (not previously worn while handling

amplified PCR products or used during sample preparation) and

clean gloves when preparing samples for PCR amplification.

• Change gloves whenever you suspect that they are

contaminated.

• Open and close all sample tubes and reaction plates carefully.

Try not to splash or spray PCR samples.

• Keep reactions and components capped as much as possible.

• Use positive-displacement or aerosol-resistant pipette tips.

• Clean lab benches and equipment periodically with freshly

diluted 10% bleach solution.

DRAFT

December 23, 2005 12:24 pm, Intro-Chem.fm

10 TaqMan MicroRNA Assays Protocol

Procedural Overview

Perform reverse transcription

65 min

15 min

90 min

Prepare the PCR reaction plate

Run the PCR reaction plate

Analyze results

(2 min)

Prepare total RNA

Thermal cycler Real-Time PCR Instruments

123456789101112

Optical 96-Well

Thermal Cycling Plate

tem

Create and set up a plate document

SDS software

Optical 384-Well

Thermal Cycling Plate

Applied Biosystems

7900HT Fast / 7300 / 7500 / 7500 Fast

Real-Time PCR System

DRAFT

December 23, 2005 12:24 pm, Assays.fm

TaqMan MicroRNA Assays Protocol 11

Using Individual Assays

Section 2 Using Individual Assays

Reverse Transcription

Overview

Synthesize single-stranded cDNA from total RNA samples using the

TaqMan

MicroRNA Reverse Transcription Kit. The process

involves the following procedures:

1. Preparing the RT master mix

2. Preparing the RT reaction plate

3. Performing reverse transcription

IMPORTANT!

TaqMan MicroRNA Assays are specifically optimized

to work with the MuLV Reverse Transcriptase contained in the

TaqMan MicroRNA Reverse Transcription Kit. Applied Biosystems

cannot guarantee assay performance if you use other reverse

transcriptase enzymes.

RNA Template

Guidelines

For the optimal performance of the TaqMan MicroRNA Reverse

Transcription Kit and of TaqMan MicroRNA Assays, Applied

Biosystems recommends using RNA with the following

characteristics:

• Free of inhibitors of reverse transcription and PCR

• Dissolved in PCR-compatible buffer

• Free of RNase activity

• Nondenatured

IMPORTANT!

Do not denature the RNA. Denaturation of the

RNA may reduce the yield of cDNA for some miRNA targets.

Per Reaction

Input Amount of

Total R N A

Use 1 to 10 ng of total RNA per 15- µL RT reaction.

DRAFT

December 23, 2005 12:24 pm, Assays.fm

12 TaqMan MicroRNA Assays Protocol

Preparing the RT

Reaction Master

Mix

Prepare RT master mix using the TaqMan MicroRNA Reverse

Transcription Kit components before preparing the reaction.

CHEMICAL HAZARD. 10✕ RT Buffer may

cause eye, skin, and respiratory tract irritation. Read the MSDS, and

follow the handling instructions. Wear appropriate protective

eyewear, clothing, and gloves.

To prepare RT master mix:

1. Allow the kit components to thaw on ice.

2. In a polypropylene tube, prepare the RT master mix by

scaling the volumes listed below to the desired number of RT

reactions. Applied Biosystems recommends adding 10 to

20% overage to account for pipetting losses.

IMPORTANT!

This procedure assumes that you are

quantifying miRNA from a single total RNA sample.

3. Mix gently. Centrifuge to bring solution to the bottom of the

tube.

4. Place the RT master mix on ice until you prepare the

microRNA reaction.

Component

Master Mix

Volume/

15-µL Reaction

100mM dNTPs (with dTTP) 0.15

MultiScribe

™

Reverse Transcriptase,

50 U/µL

1.00

10✕ Reverse Transcription Buffer 1.50

RNase Inhibitor, 20U/µL0.19

Nuclease-free water 4.16

Tota l 7 .0 0

a. Each 15-µL RT reaction consists of 7 µL master mix, 3 µL

primer, and 5 µL RNA sample.

DRAFT

December 23, 2005 12:24 pm, Assays.fm

Reverse Transcription

TaqMan MicroRNA Assays Protocol 13

Using Individual Assays

Preparing the RT

Reaction

To prepare the RT reaction:

1. For each 15-µL RT reaction, combine RT master mix (from

step 2 on page 12) with total RNA in the ratio of:

7 µL RT master mix

5 µL total RNA

For example, combine 7.7 µL of RT master mix with 5.5 µL

of total RNA. Remember to include the same proportion of

excess volume of total RNA that you did for the RT master

mix. In this example, a 10% excess volume was included for

both RT master mix and total RNA.

Note:

Applied Biosystems recommends that you use 1 to

10 ng of total RNA per reaction.

2. Mix gently. Centrifuge to bring the solution to the bottom of

the tube.

IMPORTANT!

Do not exceed 2000 RPM or 5 minutes when

centrifuging.

3. Before opening the RT Primer tubes, thaw the tubes on ice

and mix by vortexing, then centrifuge them.

4. For each 15-µL RT reaction, dispense 12.0 µL of RT master

mix containing total RNA (from step 1 on page 13) into a

0.2-mL polypropylene reaction tube. (This is the RT reaction

tube.)

Note:

Alternatively, you may dispense into a single well of a

96-well reaction plate.

5. Transfer 3 µL of RT primer (tube labeled RT Primer) from

each assay set into the corresponding RT reaction tube or

plate well.

6. Seal the tube and mix gently. Centrifuge to bring solution to

the bottom of the tube.

7. Incubate the tube on ice for 5 min and keep on ice until you

are ready to load the thermal cycler.

DRAFT

December 23, 2005 12:24 pm, Assays.fm

14 TaqMan MicroRNA Assays Protocol

Performing

Reverse

Transcription

PCR Amplification

Overview

During the target amplification step, the AmpliTaq

Gold DNA

polymerase amplifies target cDNA synthesized from the RNA

sample, using sequence-specific primers from the TaqMan Assay

Plates.

IMPORTANT!

You must perform the PCR step on a Real-Time PCR

system. Traditional thermal cyclers cannot be used because they

cannot detect and record the fluorescent signals generated by the

cleavage of TaqMan probes.

PCR Process

Performing the PCR step requires the following procedures:

1. Preparing the reaction plate

2. Setting up the plate document

3. Running the plate

To perform reverse transcription:

1. Leaving the thermal cycler in the 9600 Emulation mode

(default), use the following parameter values to program the

thermal cycler:

2. Set the reaction volume to 15.0 µL.

3. Load the reaction tube or plate into the thermal cycler.

4. Start the reverse transciption run.

Step Type

Time

(min)

Temperature

( °C)

HOLD 30 16

HOLD 30 42

HOLD 5 85

HOLD ∞ 4

DRAFT

December 23, 2005 12:24 pm, Assays.fm

PCR Amplification

TaqMan MicroRNA Assays Protocol 15

Using Individual Assays

Reagent

Preparation

Guidelines

Following these guidelines ensures optimal PCR performance:

• Keep all TaqMan MicroRNA Assays protected from light, in the

freezer, until you are ready to use them. Excessive exposure to

light may affect the fluorescent probes.

• Prior to use, mix the TaqMan Universal PCR Master Mix

thoroughly by swirling the bottle.

• Prepare the PCR reaction mix before transferring to the reaction

plate for thermal cycling and fluorescence analysis.

PCR Reaction

Components

Applied Biosystems recommends performing four PCR replicates

per RT reaction. The recommended reaction volume is 20 µL.

Prepare the plate so that each PCR reaction contains the components

as listed in the following table.

CHEMICAL HAZARD. TaqMan 2✕

Universal PCR Master Mix, No AmpErase UNG may cause eye

and skin irritation. Exposure may cause discomfort if swallowed or

inhaled. Read the MSDS, and follow the handling instructions. Wear

appropriate protective eyewear, clothing, and gloves..

Component

Volume (µL) /

20-µL Reaction

TaqMan MicroRNA Assay (20✕)1.00

Product from RT reaction (Minimum 1:15 Dilution) 1.33

TaqM a n 2✕ Universal PCR Master Mix, No

AmpErase UNG

a. For optimal performance of TaqMan MicroRNA Assays, Applied

Biosystems strongly recommends that you use Applied Biosystems

TaqM a n 2✕ Universal PCR Master Mix, No AmpErase UNG.

10.00

Nuclease-free water 7.67

Total Volume

DRAFT

December 23, 2005 12:24 pm, Assays.fm

16 TaqMan MicroRNA Assays Protocol

Preparing the

PCR Reaction

Plate

Applied Biosystems recommends performing four replicates of each

20-µL reaction.

IMPORTANT!

The following procedure assumes that you are testing

one individual assay.

CHEMICAL HAZARD. TaqMan 2✕

Universal PCR Master Mix, No AmpErase UNG may cause eye

and skin irritation. Exposure may cause discomfort if swallowed or

inhaled. Read the MSDS, and follow the handling instructions. Wear

appropriate protective eyewear, clothing, and gloves.

To prepare the PCR reaction:

1. Scale the volumes listed below to the appropriate number of

RT reactions. Applied Biosystems recommends including

four replicates per RT reaction. Prepare on ice.

2. Mix gently. Centrifuge to bring solution to the bottom of the

tube.

3. Add 17.67 µL of the PCR master mix/water mixture per

20-µL PCR reaction into a polypropylene tube (the PCR

reaction tube), as shown in the following example.

Reagent

Master Mix Volume

for One 20-µL

Reaction

TaqM a n 2✕ Universal PCR Master Mix,

No AmpErase UNG

10.00

Nuclease-free water 7.67

Total Volume 17.67

Volume for One 20-µL

Reaction

Example: Volume for 4 replicates

a. Calculation includes 12.5% extra volume to account for

pipetting losses. Keep this extra volume proportional with the

extra volume in the next two steps.

17.67

17.67 µL x 4 replicates = 70.68 µL +

8.8 µLexcess = 79.48 µL

DRAFT

December 23, 2005 12:24 pm, Assays.fm

PCR Amplification

TaqMan MicroRNA Assays Protocol 17

Using Individual Assays

Setting Up the

Plate Document

Refer to the appropriate instrument user guide for instructions on

how to configure the plate document.

• Applied Biosystems 7900HT Fast Real-Time PCR System and

SDS Enterprise Database User Guide (PN 4351684)

4. Transfer 1.0 µL of 20✕ TaqMan MicroRNA Assay mix

(labeled Real Time) into the PCR Reaction tube, as shown in

the following example.

5. Transfer 1.33 µL of the RT product from the RT reaction

tube into the PCR reaction tube, as shown in the following

example.

6. Mix gently. Centrifuge to bring solution to the bottom of the

plate.

7. Prepare the PCR reaction plate by dispensing 20 µL of the

complete PCR master mix (including primer and RT

product) into each of four wells.

8. Seal the plate with an optical adhesive cover, then centrifuge

the plate briefly to spin down the contents and eliminate any

air bubbles.

To prepare the PCR reaction: (continued)

Volume for One 20-µL

Reaction

Example: Volume for 4 replicates

1.0 1.0 µL x 4 replicates = 4 µL + 0.5 µL

excess = 4.5 µL

a. Calculation includes 12.5% extra volume to account for

pipetting losses.

Volume for One 20-µL

Reaction

Example: Volume for 4

replicates

1.33 1.33 µL x 4 replicates = 5.32 µL

+ 0.68 µL excess = 6.0 µL

a. Calculation includes 12.5% extra volume to account for

pipetting losses.

DRAFT

December 23, 2005 12:24 pm, Assays.fm

18 TaqMan MicroRNA Assays Protocol

• Applied Biosystems 7300/7500/7500 Fast Real-Time PCR

System Absolute Quantification Getting Started Guide

(PN 4347825)

When creating plate documents, use the following parameters:

Running the Plate

Refer to the following documents for detailed instructions on loading

and running the PCR plates:

• Applied Biosystems 7900HT Fast Real-Time PCR System and

SDS Enterprise Database User Guide (PN 4351684)

Parameter Value

Run Mode 9600 emulation (Default)

Sample Volume 20 µL

Thermal Cycling

Parameters

Auto Increment

Settings

Accept default values. (Default is 0.)

Ramp Rate

Settings

Accept default values. (Default is Standard.)

Data Collection Accept default values. (Default is 60 °C.)

Step

AmpliTaq

Gold

Enzyme

Activation

PCR

HOLD CYCLE (40 cycles)

Denature Anneal/

Extend

Time 10 min 15 sec 60 sec

Temp

( °C)

95 95 60

DRAFT

December 23, 2005 12:24 pm, Assays.fm

PCR Amplification

TaqMan MicroRNA Assays Protocol 19

Using Individual Assays

• Applied Biosystems 7300/7500/7500 Fast Real-Time PCR

System Absolute Quantification Getting Started Guide

(PN 4347825)

To run the plate:

1. In the SDS software, open the plate document that

corresponds to the reaction plate.

2. Load the reaction plate into the instrument.

3. Start the run.

DRAFT

December 23, 2005 12:24 pm, Assays.fm

20 TaqMan MicroRNA Assays Protocol

DRAFT

December 22, 2005 10:17 am, Analysis.fm

TaqMan MicroRNA Assays Protocol 21

Analyzing Data

Section 3 Analyzing Data

Refer to the appropriate instrument user guide for instructions on

how to analyze your data.

General Process

The general process for analyzing the data from gene expression

assays involves the following procedures:

1. View the amplification plots.

2. Set the baseline and threshold values.

Tool s f o r

Analyzing

TaqMan

MicroRNA Assay

Results

Assay Normalization with TaqMan® Endogenous Controls

Using the comparative C

method, you can use endogenous controls

to normalize the expression levels of target genes by correcting

differences in the amount of cDNA loaded into PCR reactions.

To normalize human total RNA samples, an appropriate

constitutively expressed endogenous control must be selected.

Common mRNA control transcripts are available as TaqMan

Endogenous Controls, but must be validated for the individual

researcher’s samples. More information about TaqMan Endogenous

controls is available on the Applied Biosystems Web site.

DRAFT

December 22, 2005 10:17 am, Analysis.fm

22 TaqMan MicroRNA Assays Protocol

Resources for

Data Analysis

Refer to the following documents for more information about

analyzing your data:

• Applied Biosystems 7900HT Fast Real-Time PCR System and

SDS Enterprise Database User Guide (PN 4351684)

• Applied Biosystems 7300/7500/7500 Fast Real-Time PCR

Systems Absolute Quantification Getting Started Guide

(PN 4347825)

• Livak and Schmittgen, 2001 – Provides the derivation,

assumptions, and applications of the 2

−∆∆C

method and

variations for analyzing the relative changes in gene expression

from Real-Time quantitative PCR experiments.

• Real-Time PCR Systems Chemistry Guide (Chapter 3)

(PN 4348358)

DRAFT

December 22, 2005 10:17 am, Analysis.fm

Section 3 Analyzing Data

TaqMan MicroRNA Assays Protocol 23

Analyzing Data

Bibliography

Afonina, I., Zivarts, M., Kutyavin, I., et al. 1997. Efficient priming of

PCR with short oligonucleotides conjugated to a minor groove

binder. Nucleic Acids Res. 25:2657–2660.

Bartel, D. P. 2004. MicroRNAs: Genomics, Biogenesis, Mechanism,

and Function. Cell 116:281-287.

Förster, V. T. 1948. Zwischenmolekulare Energiewanderung und

Fluoreszenz. Annals of Physics (Leipzig) 2:55–75.

Kim., J., Krichevsky, A., Grad, Y., Hayes, G.D., Kosik, K.S., Church,

G.M., and Gary Ruvkun. 2004. Identification of many microRNAs

that copurify with polyribosomes in mammalian neurons. PNAS

101:360—365.

Kutyavin, I.V., Lukhtanov, E.A., Gamper, H.B., and Meyer, R.B.

1997. Oligonucleotides with conjugated dihydropyrroloindole

tripeptides: base composition and backbone effects on hybridization.

Nucleic Acids Res. 25:3718–3723.

Kwok, S. and Higuchi, R. 1989. Avoiding false positives with PCR.

Nature 339:237–238.

Lakowicz, J.R. 1983. Energy Transfer. In Principles of Fluorescence

Spectroscopy, New York: Plenum Press 303–339.

Lim, L.P., Lau, N.C., Weinstein, E.G., Abdelhakim, A., Yekta, S.,

Rhoades, M.W., Burge, C.B., and David P. Bartel. 2003. The

microRNAs of Caenorhabditis elegans. Genes & Dev. 17: 991–1008.

Livak, K.J. and Schmittgen, T.D. 2001. Analysis of relative gene

expression data using real-time quantitative PCR and the 2

−∆∆C

method. Methods 25:402–408.

Longo, M.C., Berninger, M.S., and Hartley, J.L. 1990. Use of uracil

DNA glycosylase to control carry-over contamination in polymerase

chain reactions. Gene 93:125–128.

Mullis, K.B. and Faloona, F.A. 1987. Specific synthesis of DNA in

vitro via a polymerase-catalyzed chain reaction. Methods Enzymol.

155:335–350.

Saiki, R.K., Scharf, S., Faloona, F., et al. 1985. Enzymatic

amplification of β-globin genomic sequences and restriction site

analysis for diagnosis of sickle cell anemia. Science 230:1350–1354.

DRAFT

December 22, 2005 10:17 am, Analysis.fm

24 TaqMan MicroRNA Assays Protocol

Worldwide Sales and Support

Applied Biosystems vast distribution and

service network, composed of highly trained

support and applications personnel,

reaches 150 countries on six continents.

For sales office locations and technical support,

please call our local office or refer to our

Web site at www.appliedbiosystems.com.

Applera is committed to providing the

world’s leading technology and information

for life scientists. Applera Corporation

consists of the Applied Biosystems and

Celera Genomics businesses.

Headquarters

850 Lincoln Centre Drive

Foster City, CA 94404 USA

Phone: +1 650.638.5800

Toll Free (In North America): +1 800.345.5224

Fax: +1 650.638.5884

www.appliedbiosystems.com

01/2006

Part Number 4364031 Rev. B