Pre-miR™ miRNA Precursor Starter Kit Protocol (PN 4381862C)

Pre-miR™ miRNA Precursor Starter Kit

Part Number AM1540

Cov_Pre-miR miRNA Starter.fm Page 1 Monday, August 23, 2010 2:37 PM


(Part Number AM1540)

Protocol

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

A. Background and Product DescriptionB. Pre-miR™ miRNA Precursor Starter Kit Components and Storage ConditionsC. Required Materials Not Provided with the KitD. Related Products Available from Applied Biosystems

II. Pre-miR™ miRNA Precursor Transfection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

A. Transfection: Traditional and Reverse Transfection MethodsB. Reverse Transfection Procedure

III. Monitoring Pre-miR™ hsa-miR-1 miRNA Precursor Activity . . . . . . . . . . . . . . 11

A. RNA Isolation, Reverse Transcription, and Real-Time PCR RecommendationsB. Evaluating the Effectiveness of the Transfection ConditionsC. Optimizing Transfection Conditions

IV. Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

A. Problems with RT and/or PCRB. No Detectable Pre-miR™ hsa-miR-1 miRNA Precursor ActivityC. Transfection Causes Extensive Cell DeathD. Experiments Lack Reproducibility

V. Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

A. Traditional “Pre-plating” Transfection ProcedureB. ReferencesC. Quality ControlD. Safety Information

P/N 4381862 Revision C Revision Date: August 23, 2010

For research use only. Not for use in diagnostic procedures. By use of this product, you accept the termsand conditions of all applicable Limited Label Licenses. For statement(s) and/or disclaimer(s) applicable to thisproduct, see below.

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Literature Citation: When describing a procedure for publication using this product, please refer to it as thePre-miR™ miRNA Precursor Starter Kit.

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The Pre-miR™ miRNA Precursor Starter Kit contains Licensed Probe. Use of this product is covered by USpatent claims and patent claims outside the US. The purchase of this product includes a limited, non-transfer-able immunity from suit under the foregoing patent claims for using only this amount of product for the pur-chaser's own internal research. Separate purchase of an Authorized 5' Nuclease Core Kit would convey rightsunder the applicable claims of US Patents, and corresponding patent claims outside the United States, whichclaim 5' nuclease methods. No right under any other patent claim and no right to perform commercial servicesof any kind, including without limitation reporting the results of purchaser's activities for a fee or other com-mercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research useonly. Diagnostic uses under Roche patents require a separate license from Roche. Further information on pur-chasing licenses may be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln CentreDrive, Foster City, California 94404, USA.

Trademarks: Applied Biosystems, AB (Design), Ambion, MicroAmp, RETROscript, RNaseZap, RNase-Alert, and RNAqueous are registered trademarks, and mirVana, MagMAX, siPORT, NeoFX, and Pre-miR aretrademarks of Applied Biosystems Inc. or its subsidiaries in the US and/or certain other countries. All othertrademarks are the sole property of their respective owners.

© 2008, 2010 Ambion, Inc. All Rights Reserved.

I.A. Background and Product Description

Introduction

1

I. Introduction

A. Background and Product Description

The Ambion® Pre-miR™ miRNA Precursor Starter Kit is a collection ofreagents and protocols designed to provide an introduction tomicroRNA (miRNA) experiments, and facilitate optimization of trans-fection conditions for delivery of miRNA mimics. The kit includeseverything needed to demonstrate down-regulation of thewidely-expressed twinfilin-1(PTK9) mRNA by transfection of a syn-thetic miR-1 miRNA precursor into adherent human cultured cells. Anontargeting negative control miRNA precursor and a TaqMan®real-time PCR primer/probe set are provided for detection of PTK9down-regulation using real-time reverse transcription (RT)-PCR.

Background on microRNA

(miRNA)

miRNA is a class of endogenous 17–24 nt noncoding RNAs that areimportant regulators of gene expression in many organisms. Like siRNAs,miRNAs target individual mRNAs via base-pairing interactions mediatedby the RNA-induced silencing complex (RISC) pathway. In contrast tosiRNAs, many animal miRNAs appear to effect silencing by repressingtranslation rather than by inducing cleavage of cognate mRNAs (Bartel2004, Carrington 2003). Evidence is accumulating, however, that someanimal miRNAs can reduce the level of their target mRNAs (as do themajority of plant miRNAs), so that their effects can be monitored usingmethods associated with gene expression analysis such as real-timeRT-PCR and array analysis (Lim 2005, Schramke 2005, and Yekta 2004).

Pre-miR™ miRNA

Precursors

Ambion Pre-miR™ miRNA Precursors are small, chemically modifieddouble-stranded RNA molecules designed to mimic endogenous maturemiRNA molecules. They are similar, but not identical, to siRNAs. Thedesign and chemical modification of Pre-miR miRNA Precursors ensurethat the correct strand, representing the desired mature miRNA, is takenup into the RNA-induced silencing complex (RISC)-analogous complexresponsible for miRNA activity. In contrast to miRNA expression vec-tors, these synthetic molecules can be used in dose response studies dueto their introduction directly into the cell by transfection or electropora-tion. Because of their small size, they are easier to transfect than plasmidvectors and can be delivered using conditions similar to those used forsiRNAs. They enable detailed study of miRNA biological effects viagain-of-function experiments. Examples of experimental uses include:

• miRNA target site identification and validation

• Screening for miRNAs that regulate the expression of a gene

• Screening for miRNAs that affect a cellular process


I.A. Background and Product Description2

The Pre-miR hsa-miR-1

miRNA Precursor and

nontargeting control

supplied with the kit

The Pre-miR miRNA Precursor Starter Kit is supplied with a Pre-miRhsa-miR-1 miRNA Precursor and a nontargeting negative controlPre-miR miRNA Precursor (Pre-miR Negative Control #1). Endoge-nous miR-1 has been shown to negatively regulate PTK9 mRNA in cul-tured cells. This is thought to occur through a base-pairing interactionin the 3' untranslated region of the PTK9 message that results in mRNAcleavage (Lim 2005). When transfected into human and mouse celllines, Pre-miR hsa-miR-1 miRNA Precursor mimics endogenous miR-1miRNA and reduces the expression of PTK9.

NOTE

The Pre-miR hsa-miR-1 miRNA Precursor supplied with the kit will reduce the

expression of PTK9 when transfected into human or mouse cultured cells.

However, the supplied PTK9 TaqMan® Gene Expression Assay can only be

used for detection of cDNA synthesized from human PTK9 mRNA. To detect

Pre-miR hsa-miR-1 miRNA Precursor activity in mouse cultured cells, pur-

chase a mouse-specific PTK9 TaqMan Gene Expression Assay; we recom-

mend Assay ID: Mm01598980-g1.

The Pre-miR Negative Control #1 miRNA Precursor is a nontargetingsequence that bears no homology to the sequences of human, mouse, orrat transcripts. Empirically it has been shown to have no effect on PTK9mRNA levels or cell proliferation, survival, or morphology.

Delivery of Pre-miR™

miRNA Precursor

Transfection agent

The Pre-miR miRNA Precursor Starter Kit is supplied with thelipid-based siPORT™ NeoFX™ transfection agent, for transfection ofPre-miR miRNA Precursors (hsa-miR-1 and Pre-miR Negative Control#1) into adherent cultured cells. siPORT NeoFX transfection agentfacilitates small nucleic acid transfection in a broad range of cell typeswith low cytotoxicity, and high efficiency and reproducibility.

Transfection method

With siPORT NeoFX transfection agent, we recommend the rapid,effective transfection method called reverse transfection. In reversetransfection, cells are mixed with transfection complexes as they adhereto a plate after trypsinization (Figure 2 on page 7). This methodbypasses several steps of the traditional “pre-plating” transfectionmethod, making it faster and easier. A Procedure for traditional trans-fection is provided in section V.A on page 20.

Cell type

Adherent HeLa cells are a good choice for use with this kit because theycan be reliably transfected with siPORT NeoFX transfection agent withminimal toxicity following these instructions.

I.B. Pre-miR™ miRNA Precursor Starter Kit Components and Storage Conditions

Introduction

3

Detection of Pre-miR™

hsa-miR-1 miRNA Precursor

activity

A 20X PTK9 TaqMan® Gene Expression Assay, Hs00702289_s1, whichincludes PCR primers and a TaqMan probe, is included to detect reduc-tion of human PTK9 mRNA levels using real-time RT-PCR. (See the Noteon page 2 for information about using the kit with mouse cultured cells.)

B. Pre-miR™ miRNA Precursor Starter Kit Components and Storage

Conditions

C. Required Materials Not Provided with the Kit

Cell culture material and

equipment

• Adherent mammalian cultured cells derived from humanThis kit was developed using HeLa cells primarily because they are areadily available cell line that can be reliably transfected usingsiPORT NeoFX transfection agent.

NOTE

The PCR primers and TaqMan® probe in the PTK9 TaqMan Assay pro-

vided for evaluation of your results will amplify only cDNA from human

PTK9 mRNA. The other kit reagents and procedures can be used with cul-

tured cells derived from mouse, but you must purchase a mouse-specific

PTK9 TaqMan Gene Expression Assay in order to detect PTK9 down-reg-

ulation in mouse (we recommend Applied Biosystems Assay ID:

Mm01598980_g1.)

• Opti-MEM® I Reduced-Serum Medium (Invitrogen #31985-062)• Routine tissue culture supplies and equipment

RNA isolation reagents and

equipment

Any RNA isolation method that is appropriate for cultured mammaliancells can be used to obtain RNA for RT-PCR analysis. We recommendthe following kits from Ambion:• Ambion MagMAX™-96 Total RNA Isolation Kit, P/N AM1830• Ambion RNAqueous®-96 High Throughput, 96-well RNA Isolation

Kit, P/N AM1920

Amount Component Storage

5 nmol Pre-miR™ hsa-miR-1 miRNA Precursor –20°C

5 nmol Pre-miR™ Negative Control #1 –20°C

250 μL Hs00702289_s120X PTK9 (PCR Primer/Probe Set)

–20°C

400 μL siPORT™ NeoFX™ Transfection Agent*

* Keep the tube of siPORT NeoFX tightly closed to prevent evaporation.

4°C

1.75 mL Nuclease-free Water any temp†

† Store Nuclease-free Water at –20°C, 4°C or room temp.


I.D. Related Products Available from Applied Biosystems4

RT-PCR materials and

equipment

We recommend using a two-step RT-PCR strategy to evaluate Pre-miRhsa-miR-1 miRNA Precursor activity. For normalization of thereal-time PCR, we recommend evaluating the expression of an endoge-nous control in parallel.

Reverse transcription reagents:

Any random-primed method for reverse transcription can be used togenerate cDNA for the PTK9 TaqMan Assay. We recommend the fol-lowing products from Applied Biosystems:• Ambion RETROscript® Kit, P/N AM1710• Applied Biosystems High Capacity cDNA Reverse Transcription

Kit, P/N 4322171, 4368813, 4374967, 4368814, 4374966

Real-time PCR reagents and equipment:

• We recommend Applied Biosystems TaqMan® PCR Universal PCRMaster Mix (P/N 4324018).

• We recommend any of Applied Biosystems Real-Time PCR Sys-tems, for example the StepOne™, StepOnePlus™, 7900, 7900HT, or7500 Real-Time PCR Systems.

TaqMan® Gene Expression Assay for an endogenous control:

To normalize gene expression data obtained with the PTK9 TaqManAssay, you will need to run parallel reactions to evaluate the expressionlevels of an endogenous control such as 18S rRNA or cyclophilin. Werecommend the following Applied Biosystems TaqMan Gene Expres-sion Assays for eukaryotic 18S rRNA, Assay ID: Hs99999901_s1

Plasticware for RNA isolation and real-time RT-PCR such as:

• Nuclease-free microcentrifuge tubes (e.g., Ambion RNase-freeMicrofuge Tubes, P/N AM12300, Non-Stick RNase-free MicrofugeTubes, P/N AM12350)

• Barrier tips for pipettors (Ambion P/N AM12635, AM12640,AM12645, AM12650, AM12665)

• Applied Biosystems MicroAmp™ Optical 96-Well Reaction Platesand Optical Adhesive Films and Optical Caps

D. Related Products Available from Applied Biosystems

siPORT™ NeoFX™

Transfection AgentP/N AM4510, AM4511

siPORT NeoFX Transfection Agent was developed to streamline siRNA trans-fection procedures, cutting time and increasing reproducibility. This novellipid-based formulation can be used to efficiently transfect adherent cellswhile subculturing, without increased cytotoxicity. This reagent is compatiblewith a wide range of cell lines and experimental designs, includinghigh-throughput applications.

siPORT™ Amine Transfection

AgentP/N AM4502, AM4503

siPORT Amine Transfection Agent is an easy-to-use proprietary blend ofpolyamines that delivers siRNA into mammalian cells with minimal cytotoxicity.

I.D. Related Products Available from Applied Biosystems

Introduction

5

Pre-miR™ miRNA PrecursorsP/N AM17100, AM17101,

AM17103

mirVana™ Pre-miR™ miRNA Precursors are small, chemically modified dou-ble-stranded RNA molecules designed to mimic endogenous mature miRNAmolecules. These ready-to-use miRNA mimics can be introduced into cellsusing transfection or electroporation parameters identical to those used forsiRNAs and enable detailed study of miRNA biological effects viagain-of-function experiments. Pre-miR miRNA Precursors are available for allmiRNAs listed in the miR Base database and custom design is available.

Anti-miR™ miRNA InhibitorsP/N AM17110, AM17111

mirVana™ Anti-miR™ miRNA Inhibitors are chemically modified, sin-gle-stranded nucleic acids designed to specifically bind to and inhibit endog-enous microRNA (miRNA) molecules. These ready-to-use inhibitors can beintroduced into cells using transfection or electroporation parameters similarto those used for siRNAs, and enable miRNA functional analysis bydown-regulation of miRNA activity.

MagMAX™-96 Total RNA

Isolation KitP/N AM1830

The MagMAX™-96 Total RNA Isolation Kit is a magnetic bead based totalRNA purification system designed for rapid high throughput processing ofcells in 96 well plates. High yield and high quality total RNA can be obtainedfrom 100 to 500,000 cultured eukaryotic cells. The kit can also be used fortotal RNA isolation from small tissue samples.

RNAqueous®-96 KitP/N AM1920

The RNAqueous-96 Kit is designed for high throughput, 96 well sample pro-cessing for isolation of total RNA from multiple samples and very small sam-ples. The procedure utilizes an RNA-binding glass-fiber filter to provide highyields of intact RNA without using organic solvents (such as phenol). Anoptional on-the-filter DNase treatment can be performed to ensure removalof genomic DNA for RT-PCR applications.

High Capacity cDNA Reverse

Transcription KitP/N 4322171, 4368813, 4374967,

4368814, 4374966

Applied Biosystems High Capacity cDNA Reverse Transcription Kit (for-merly the High Capacity cDNA Archive Kit) delivers extremely high-quality,single-stranded cDNA from total RNA. It contains all components necessaryfor the quantitative conversion of 0.02–2 μg of total RNA to cDNA in a20 μL reaction.

RETROscript® KitP/N AM1710

First strand cDNA synthesis kit for RT-PCR. When purchased with Super-Taq™ thermostable DNA polymerase, this kit provides reagents, controls andprotocols for reverse transcription and PCR. Both oligo(dT) and randomprimers for cDNA priming are included, as is RNase inhibitor.

TaqMan® Universal PCR

Master MixSee web or print catalog for P/Ns

Applied Biosystems TaqMan® Universal PCR Master Mix combines the com-ponents needed for the fluorogenic 5’ nuclease assay in one easy-to-use pre-mix. The proprietary buffer components and stabilizers are optimized toenhance reaction performance across all sample types. TaqMan UniversalPCR Master Mix is available with and without uracil-DNA glycosylase,UNG, which prevents carry-over contamination from previous PCRs.


II.A. Transfection: Traditional and Reverse Transfection Methods6

II. Pre-miR™ miRNA Precursor Transfection

A. Transfection: Traditional and Reverse Transfection Methods

Transfection overview Delivery of Pre-miR miRNA Precursors into cultured cells requires theuse of chemical transfection agents. Lipid-based transfection agents,such as siPORT™ NeoFX™ Transfection Agent, facilitate transfection bycomplexing into aggregates with the negatively charged nucleic acids.These nucleic acid-transfection agent complexes are efficiently taken upby cells, presumably by endocytosis (Figure 1).

Traditional (Pre-plated)

transfection compared to

reverse transfection

Figure 2 shows overviews of the traditional and reverse transfectionmethods. In traditional transfection of adherent mammalian cells, thecells are pre-plated 24 hr before transfection, and cultured to recoverfrom trypsinization, grow, and adhere to the culture plate. We recom-mend traditional or pre-plated transfection as the first transfectionmethod to try with Ambion siPORT™ Amine Transfection Agent.Reverse transfection is a time-saving, effective alternative in which cellsare transfected as they are subcultured. Compared to the traditionalpre-plating method, equivalent or improved transfection efficiency isseen for many of the cell types tested at Ambion. In addition, the reversetransfection process is an entire day shorter than traditional transfec-tion. Because cells are in suspension, a larger amount of cell surface isexposed to transfection agent/nucleic acid complexes, and this isthought to contribute to the improved transfection efficiency. We rec-ommend reverse transfection as the first transfection method to try withAmbion siPORT NeoFX Transfection Agent.

Figure 1. Chemical Transfection of miRNA into Cells.

• Incubate Pre-miR™ miRNA Precursor with transfection agent to form a complex• Mix Pre-miR miRNA Precursor/transfection agent complexes with cells• Transfection agent facilitates uptake of Pre-miR miRNA Precursor into the cytoplasm

Pre-miR™ miRNA Precursor/transfection agent complex

cytoplasm

cellularmembrane

nucleus

II.B. Reverse Transfection Procedure

Pre-miR™ miRNA Precursor Transfection

7

Because siPORT NeoFX Transfection Agent is supplied with this kit,the main procedure is written for reverse transfection; however, we alsoinclude a procedure for traditional pre-plated transfection insection V.A starting on page 20.

B. Reverse Transfection Procedure

1. Preparation and planning a. Resuspend Pre-miR miRNA Precursors to 6.25 μM

Add 800 μL of Nuclease-free Water to each tube containing 5 nmolof Pre-miR miRNA Precursor (both the Pre-miR hsa-miR-1 miRNAPrecursor and Pre-miR Negative Control #1) for a final concentra-tion of 6.25 μM. Mix thoroughly and store the Pre-miR miRNA Precursor solutionsat –20°C for up to 6 months.

b. Bring siPORT™ NeoFX™ Transfection Agent and Opti-MEM I

medium to room temp before use in step 3 on page 8.

c. Plan the total number of transfections in your experiment

For each set of transfection conditions, plan to include 2–3 replicatetransfections with each of the following (i.e., a total of 6–9 transfec-tions for each set of transfection conditions):• Pre-miR hsa-miR-1 miRNA Precursor• Pre-miR Negative Control #1

Figure 2. Comparison of Traditional (Pre-plated) and Reverse Transfection Methods

Mix transfectionagent with nucleic acid

Add transfectioncomplex to plated cells

Dilute transfectionagent

Harvest cellsPlate cellsand grow for 24 hr

Aliquot nucleic acidto transfect

Incubate and assay cells

Overlay with cells

Pre-platedTransfection

Day 1plate cells

Day 2transfect

Day 4assay

ReverseTransfection

Day 1transfect

Day 3assay


II.B. Reverse Transfection Procedure8

IMPORTANT

• Nontransfected control: cells that are mock-transfected withOpti-MEM I medium, but no transfection agent and no Pre-miRmiRNA Precursor.

NOTE

In this section, suggested initial transfection conditions are listed first and

reagent quantities for transfection optimization experiments follow. A work-

flow for how to optimize transfection conditions is provided in section III.C.

Optimizing Transfection Conditions starting on page 14.

2. Prepare cells a. Trypsinize adherent cells.

Trypsinize healthy, growing, adherent cells using your routine pro-cedure. In general, healthy cells transfect better than poorly main-tained cells. Routinely subculturing cells before they becomeovercrowded or unhealthy will improve transfection and minimizeinstability in cell lines from experiment to experiment. Informationon basic cell culture technique can be found in Culture of AnimalCells: A Manual of Basic Technique (2000) Freshney, NY:Wiley-Liss.

b. Resuspend cells in normal growth medium.

For initial experiments, resuspend cells in normal growth mediumto 1 x 105 cells/mL.

In order for cells to be transfected

before they re-adhere, it is important

to proceed immediately to

transfection (steps 3–4) after

preparing cells.

For subsequent transfection optimization experiments, we recom-mend testing from 5 x 104 to 1.5 x 105 cells/mL. To do this, firstsuspend cells at 1.5 x 105 cells/mL and then dilute a portion of thesuspension further to 1 x 105 cells/mL and 5 x 104 cells/mL.Keep the cells at 37°C until they are needed in step 4. The table below shows the volume of cells needed per well in differ-ent culture plates. Count the number of wells in your experiment,and calculate the total volume of cells you will need. Plan to prepare~5% overage to account for pipetting error.

3. Prepare Pre-miR miRNA

Precursor/siPORT NeoFX

complexes and distribute

into culture plate wells

• Briefly centrifuge the siPORT NeoFX Transfection Agent before use.• The instructions below show the reagent amounts needed for indi-

vidual reactions using the indicated size of cell culture plate. Plan toprepare master mixes of common reagents, with ~5–10% overage, tolimit the pipetting steps needed for your experiment and minimizevariability.

Amounts per transfection

Culture Plate Type

96 well 24 well 6 well

Volume of cells 80 μL 400 μL 2.4 mL

Number of cellsrecommended for first expt(range for optimization)

8 x 103

(2–20 x 103)4 x 104

(2–10 x 104)2.4 x 105

(1–5 x 105)

II.B. Reverse Transfection Procedure

Pre-miR™ miRNA Precursor Transfection

9

a. Dilute siPORT NeoFX Transfection Agent in Opti-MEM I

medium in a sterile tube.

b. Incubate the diluted transfection agent for 10 min at room

temp.

c. Dilute the 6.25 μM Pre-miR hsa-miR-1 miRNA Precursor or

Pre-miR Negative Control #1 in Opti-MEM I medium.

(Instructions for preparing a 6.25 μM Pre-miR miRNA Precursorsolution are in step 1.a on page 7.)

d. Gently mix diluted Pre-miR miRNA Precursor with diluted

siPORT NeoFX Transfection Agent.

Combine diluted siPORT NeoFX (or other transfection agent) fromstep b with the diluted Pre-miR miRNA Precursor from step c. Mixgently by pipetting up and down or flicking the tube several times.

e. Incubate at room temp for 10 min.

Incubate 10 min at room temp. Nucleic acid/siPORT NeoFX trans-fection complexes form during this incubation.

f. Dispense the transfection complexes into the empty wells of

a culture plate, and set up the nontransfected controls.

Aliquot miRNA precursor/siPORT NeoFX transfection complexesfrom step e (previous) into the wells of the culture plate followingthe volume guidelines shown below. Include 3 nontransfected con-trol wells, containing only Opti-MEM I medium (but no Pre-miRmiRNA Precursor and no transfection agent).

Amounts per transfection 96 well 24 well 6 well

siPORT NeoFX*recommended for first expt(range for optimization)

* Note that these volumes are appropriate for siPORT NeoFX, but may not be appropriate for other transfection agents. Use the manufacturer’s volume rec-ommendations for other transfection agents.

0.3(0.15–1.2

μLμL)

1.5(0.75–6

μLμL)

9(4.5–36

μLμL)

Opti-MEM I to final volume: 10 μL 50 μL 300 μL

Amounts per transfection 96 well 24 well 6 well

6.25 μM Pre-miR*recommended for first expt(range for optimization)

* We recommend initially using 50 nM final Pre-miR miRNA Precursor concen-tration (that is, the concentration in the transfection mixture at the end of step 4 on page 10), and to test 5, 20, 50, and 100 nM final Pre-miR miRNA Precursor concentration in optimization experiments.

0.8(0.08–1.6

μLμL)

4(0.4–8

μLμL)

24(2.4–48

μLμL)

Opti-MEM I to final volume: 10 μL 50 μL 300 μL

Amount per transfection 96 well 24 well 6 well

Transfection complex or Opti-MEM I (controls)

20 μL 100 μL 600 μL

siPORT™ NeoFX™ Transfection Agent in Opti-MEM I

Dilute Pre-miR™ miRNAPrecursor in Opti-MEM I

Incubate 10 minat room temp

Mix

Incubate 10 minat room temp to form

transfection complexes


II.B. Reverse Transfection Procedure10

4. Add cells to the

transfection complexes

(and control wells)

a. Transfer cells to the culture plate.

Gently mix the cells prepared in step 2 to resuspend, and pipet theminto wells of the culture plate containing Pre-miR miRNA Precur-sor/siPORT NeoFX Transfection Agent complexes or into wells setup as nontransfected controls.

b. Gently rock the transfection mixture.

Rock the plate gently back and forth to evenly distribute the cells andthe transfection complexes. Avoid swirling, as this can cause contentsto aggregate in the center of the well.

5. Incubate at 37°C for 24 hr,

then replace the culture

medium

Incubate the transfection mixture at 37°C in normal cell culture condi-tions for 24 hr. Then, replace the culture medium with fresh normalgrowth medium.

6. Check the visual

appearance of the

transfected cells

As an initial screen for severe cytotoxic effects, check the visual appear-ance of transfected cells for evidence of cell necrosis and/or apoptosisbefore investing the time to evaluate cytotoxicity and PTK9 downregu-lation in more detail.

Amount per transfection 96 well 24 well 6 well

Volume of cells per well 80 μL 400 μL 2.4 mL

Total volume (100 μL) (500 μL) (3 mL)

III.A. RNA Isolation, Reverse Transcription, and Real-Time PCR Recommendations

Monitoring Pre-miR™ hsa-miR-1 miRNA Precursor Activity

11

III. Monitoring Pre-miR™ hsa-miR-1 miRNA Precursor Activity

A. RNA Isolation, Reverse Transcription, and Real-Time PCR

Recommendations

When transfected into mammalian cells, the Pre-miR hsa-miR-1miRNA Precursor is expected to reduce the amount of PTK9 mRNA inthe cells. The Pre-miR miRNA Precursor Starter Kit is supplied with aPTK9 TaqMan® Gene Expression Assay for using real-time RT-PCR tomeasure the amount of PTK9 mRNA in human cells that have beentransfected with Pre-miR hsa-miR-1 miRNA Precursor.

NOTE

Unlike the Pre-miR hsa-miR-1 miRNA Precursor, many Pre-miR miRNA Pre-

cursors act at the translational level only, reducing the protein product of

their target gene, but not significantly affecting the quantity of target mRNA.

1. Isolate total RNA from

transfected cells

Two days after transfection, isolate total RNA from cells transfected witheach set of transfection conditions and from all control transfections.Any method that yields RNA of sufficient quality for RT-PCR is accept-able, however, we recommend the Ambion® MagMAX™-96 Total RNAIsolation Kit (P/N AM1830) or RNAqueous®-96 Kit (P/N AM1920) fortheir simple, rapid protocols and high yields of high quality RNA.

NOTE

RNAqueous-96 Kit users: the DNase treatment is optional for preparation of

RNA for use in this procedure.

2. Reverse transcribe the

RNA to produce cDNA

Reverse transcribe a portion of the RNA from each sample to producecDNA. We recommend using either the Ambion RETROscript® Kit(P/N AM1710) or Applied Biosystems High Capacity cDNA ReverseTranscription Kit (P/N 4368814, 4374966) for these reactions, but anyrandom-primed method for reverse transcription (RT) can be used. Fol-lowing are our recommendations for reverse transcription:• Use random primers for the reactions.• Use the same amount of RNA in each reaction. We recommend

reverse transcribing 100ng–2 μg of RNA.• Include a no-RT negative control that includes all the reaction com-

ponents except the reverse transcriptase. This will serve as a controlfor amplification of genomic DNA (without reverse transcriptase,the sample will not contain cDNA).

• Prepare master mixes of common reagents, with ~5–10% overage toto minimize variability.

• We suggest using a 20 μL RT reaction volume.


III.A. RNA Isolation, Reverse Transcription, and Real-Time PCR Recommendations12

3. Perform real-time PCR to

detect PTK9 and

endogenous control gene

expression

Recommended controls and replicates• Run duplicate PCRs (or more).• Include reactions to amplify an endogenous control RNA such as 18S

ribosomal RNA or cyclophilin for normalization of results. In otherwords, amplify samples from each RT using both a PTK9 TaqManAssay and a real-time PCR assay for an endogenous control gene.

• Use a sample of the no-RT negative control as template for amplifi-cation with each TaqMan Assay.

• Include a no-template control for each set of PCR primers/probeused. This negative control helps to identify DNA contamination ofthe PCR reagents.

a. Program the real-time PCR instrument.

Following are PCR cycling conditions and a suggested reactionset-up for a single reaction using Applied Biosystems TaqMan® Uni-versal PCR Master Mix, No AmpErase® UNG (P/N 4324018).(TaqMan Universal PCR Master Mix that does contain AmpEraseUNG requires an initial 2 min incubation at 50°C.)

b. Assemble the PCRs

Prepare a cocktail of common reagents, with ~5–10% overage tominimize variability.

• TaqMan Gene Expression Assays are a ready-to-use 20X mixtureof forward and reverse PCR primers and a TaqMan probe. Add0.5 μL TaqMan Assay to the PCR for a final concentration of 1X.

• We generally include 2 μL of the RT reaction (cDNA) in eachPCR. For amplification of very highly expressed endogenous con-trol messages such as 18S rRNA, however, you may have to dilutea portion of the RT 1:30, and use 2 μL of the diluted cDNA toget amplification in the linear range of detection.

• We suggest using a 20 μL PCR volume.

c. Run the thermal cycle and determine CT values for each

reaction according to the PCR instrument manufacturer’s

instructions

Stage Reps Temp Time

Initial denaturation 1 1 95°C 10 min

Amplification 2 40 95°C 15 sec

60°C 1 min

Amount Component

5 μL TaqMan® Universal PCR Master Mix, No AmpErase® UNG (2X)

0.5 μL TaqMan® Gene Expression Assay (20X)

2 μL RT Reaction (cDNA)

2.5 μL Nuclease-free Water

III.B. Evaluating the Effectiveness of the Transfection Conditions


13

B. Evaluating the Effectiveness of the Transfection Conditions

1. Calculate the normalized

CT (or ΔCT) for PTK9 from

each sample

For these calculations, use the mean CT value from the duplicate PCRs.

In real-time RT-PCR, the CT of the experimental amplicon, PTK9 inthese experiments, is inversely proportional to the quantity of the targetmRNA after normalization to the CT value for an endogenous controlgene mRNA. This normalized CT value is known as ΔCT, and for thePTK9 amplicon, it is defined as follows:

ΔCT = CT for PTK9 – CT for endogenous control

2. Calculate the percent

downregulation of PTK9

PTK9 downregulation is related to the difference in the ΔCT value forsamples transfected with Pre-miR hsa-miR-1 miRNA Precursor (ΔCTmiR-1) compared to the ΔCT value for samples transfected withPre-miR Negative Control #1 (ΔCT Neg#1). This is known as the ΔΔCt:

ΔΔCT = ΔCTmiR-1 – ΔCTNeg#1

The percent downregulation can then be determined using the relation:% downregulation = 100 – 100 X 2–ΔΔCT

3. Evaluate transfection-

associated cytotoxicity

from CT values

Transfection agents are somewhat toxic to cells. Since transfection withthe Pre-miR Negative Control #1 miRNA Precursor will not affect geneexpression, any decrease in the expression level of the endogenous con-trol in cultures transfected with the Pre-miR Negative Control #1 can beattributed to cytotoxicity. Specifically, compare the CT of the endoge-nous control amplification in cells transfected with Pre-miR NegativeControl #1(CT Neg #1) to that in nontransfected cells (CT nontrans-fected) to examine the cytotoxic effects of the transfection conditions.Transfection conditions which do not cause any cytotoxicity wouldresult in a ratio of 1, therefore, the closer this ratio is to 1, the less cyto-toxic the transfection conditions.

Using results from the endogenous control amplification: CT Neg #1 ÷ CT nontransfected = cytotoxicity factor

4. Calculating the best

transfection condition

tested

Optimal conditions for Pre-miR miRNA Precursor transfection for agiven cell type are those which simultaneously maximize the percentdownregulation of PTK9 and minimize transfection-associated cytotox-icity. A useful way to describe the balance of transfection efficiency andcytotoxicity is the term Optimal Balance Factor, or OBF. The OBF iscalculated for each transfection condition as follows:

OBF = (cytotoxicity factor) X (% downregulation)OBF = (CT Neg #1 ÷ CT nontransfected) X (100 – 100 X 2–ΔΔCT)

Typically, optimal transfection condition(s) are those which exhibithighest OBF value.


III.C. Optimizing Transfection Conditions14

C. Optimizing Transfection Conditions

When is transfection

optimization needed?

As a general guideline, we recommend optimizing transfection condi-tions so that a ≥60% reduction in PTK9 mRNA is seen, as measured byreal-time RT-PCR, with ≤25% cytotoxicity or cell death. Figure 3 onpage 14 shows an overview of the transfection optimization strategydescribed in this section.

Optimized transfection is a

balance of cytotoxicity and

Pre-miR miRNA Precursor

activity

The goal of transfection optimization is to identify the conditions thatwill provide good Pre-miR miRNA Precursor activity while minimizingtransfection-induced cytotoxicity for the particular cell type. Typically,conditions which improve Pre-miR miRNA Precursor activity (e.g.,increase in amount of transfection agent) also result in increased cyto-toxicity. Therefore, both activity of the transfected Pre-miR miRNAPrecursor and cytotoxicity must be considered when interpreting opti-mization experiments—with a balance between the two representingthe ideal conditions for transfection. Once optimal conditions are estab-lished, they should be kept constant among experiments for a given celltype.

1. Choice of transfection

agent

Ambion’s siPORT NeoFX Transfection Agent is highly effective withminimal cytotoxic effects. It is useful for a wide variety of adherent cul-tured mammalian cells. However, different cell types may vary in theirresponse to a given transfection agent. Therefore, in some instancessiPORT NeoFX may not efficiently transfect a particular cell type. Inthis case, test other transfection agents such as Ambion siPORT AmineTransfection Agent (P/N AM4502, AM4503).

Figure 3. Transfection Optimization Strategy.

First, follow the procedure in section B. Reverse Transfection Procedure on page 7 to evaluate the effectiveness of initial sug-gested transfection conditions with siPORT™ NeoFX™ Transfection Agent. The goal is to achieve ≥60% PTK9 down-reg-ulation measured by real-time RT-PCR with ≤25% cytotoxicity. If these values are achieved, no further optimization isneeded. Otherwise, optimize transfection using the strategy described here.

Optimizetransfection agent

e.g., try siPORT™ Amine

siPORT™ NeoFX™(included in the kit)

If cytotoxicity encountered

Optimizecell exposure to

transfection agent

To enhance overall transfection efficiency, optimize cell density

If no cytotoxicity

Optimize transfection agent volume

To optimize down-regulationtest different

amounts of Pre-miR™miRNA Precursor

III.C. Optimizing Transfection Conditions


15

a. For Ambion siPORT Amine Transfection Agent, follow theprocedure in section V.A. Traditional “Pre-plating” TransfectionProcedure starting on page 20. For other transfection agents, you mayfollow the general procedure in section II.B. Start with a 50 nM finalconcentration of Pre-miR miRNA Precursor. Follow the transfectionagent manufacturer’s recommendations for the volume oftransfection agent to use.

b. Assay for Pre-miR hsa-miR-1 miRNA Precursor activity (PTK9down-regulation) and cytotoxicity. • If one of the transfection agents provides ≥60% PTK9 down-reg-

ulation as measured by real-time RT-PCR with ≤25% cytotoxic-ity, no further optimization is necessary.

• If the observed results do not meet these criteria, select the trans-fection agent that gave better overall results, and proceed tostep 2 below.

2. Amount of transfection

agent

The volume of transfection agent used is a critical parameter to opti-mize; too little can limit transfection, but too much can be toxic. Theoverall transfection efficiency is influenced by the amount of transfec-tion agent complexed to the Pre-miR miRNA Precursor.

a. Follow the procedure in section II.B or V.A (using 50 nM finalconcentration of Pre-miR miRNA Precursor) to test four differentvolumes of transfection agent (step II.B.3 of the reverse transfectionprocedure), following the guidelines shown in the tables foroptimization experiments.

b. Assay for Pre-miR hsa-miR-1 miRNA Precursor activity (PTK9down-regulation) and cytotoxicity. • If good PTK9 down-regulation and minimal cytotoxicity (as

defined above) are obtained, no further optimization is necessary. • If >25% cytotoxicity is observed, proceed to step 3. • If acceptable levels of cytotoxicity are obtained, but PTK9

down-regulation is insufficient, proceed to step 4.

3. Exposure time to

transfection agent

(if needed)

Although siPORT NeoFX Transfection Agent was designed to minimizecytotoxicity, exposing cells to excessive amounts of transfection agent orfor extended time periods can be detrimental to the overall health of thecell culture.

After determining the optimal volume of transfection agent for PTK9down-regulation, minimize cytotoxicity by adjusting the time that cellsare exposed to transfection complexes.

a. Replace the medium at 6 hr and 12 hr after transfection by carefullyaspirating the old medium from the well and adding fresh medium. Itis usually not necessary to wash cells.


III.C. Optimizing Transfection Conditions16

b. Re-evaluate Pre-miR hsa-miR-1 miRNA Precursor activity (PTK9down-regulation) and cytotoxicity. • If good PTK9 down-regulation and minimal cytotoxicity (as

defined above) are obtained, no further optimization is necessary. • If PTK9 down-regulation is insufficient, proceed to step 4.

4. Amount of Pre-miR

miRNA Precursor

A number of miRNAs share considerable sequence homology. The like-lihood of downregulation of a closely related target increases withPre-miR miRNA Precursor concentration. Thus, to minimize off-targetinteractions, use the lowest Pre-miR miRNA Precursor concentrationwhich shows activity in downregulating the target gene.

To optimize the activity of transfected Pre-miR miRNA Precursors, test5, 20, 50, and 100 nM (final concentration) Pre-miR miRNA Precur-sor, using the transfection agent quantity and exposure time optimizedin the experiments described above.

5. Cell density For most adherent cells, the optimal confluency for transfection is30–80%. The cell amounts listed as the range for optimization instep II.B.2 on page 8 and Table 1 on page 20 provide guidelines forseeding culture plates of different sizes to obtain 30–80% confluencyafter 24 hr of growth; these numbers are approximate because the exactnumber of cells required for seeding and transfection depends on celltype, size, and growth rate. • Use the transfection conditions optimized in the steps above, while

varying the cell plating density across the wells of the culture dish sothat cells will reach between 30–80% confluency.

• Be sure to monitor cell viability during these experiments, as cell cul-tures can become unstable at low densities.

The optimal cell plating density results in the greatest reduction inPTK9 expression without creating instability in the cell line.

IV.A. Problems with RT and/or PCR

Troubleshooting

17

IV. Troubleshooting

A. Problems with RT and/or PCR

Abnormal amplification plot

for the endogenous control

reaction

Too much template in the PCR

If signal from the endogenous control amplification is detected in theearly cycles of PCR and the CT value is <15, it is an indication that toomuch template was used in the PCR. This results in an abnormal ampli-fication plot. For amplification of very highly expressed endogenous control messagessuch as 18S rRNA, you may have to dilute a portion of the reverse tran-scription reaction 1:30, and use 2 μL of the diluted cDNA in real-timePCR for good results.

No signal RNA contains inhibitors

If PTK9 is not detected even in samples that were transfected with thePre-miR Negative Control #1, it may indicate that the RNA containsinhibitors. Samples containing minimal amounts of inhibitors mayyield successful RT-PCRs by adding less sample (and therefore lessinhibitor), to the reaction. Accomplish this by diluting samples, forexample 5- and 10-fold, and then use 2 μL in PCR.

Signal detected in the

no-template control

PCR contamination

The most likely cause of signal detection in the no-template negativecontrol reaction is contamination of PCR reagents with DNA (oftenfrom previous PCRs). In this case, repeat the PCR with fresh reagentsand freshly decontaminated pipettors. Also set up PCRs in an area iso-lated from areas used for RNA isolation and PCR product analysis.

B. No Detectable Pre-miR™ hsa-miR-1 miRNA Precursor Activity

The transfection procedure

requires optimization

You may need to optimize the transfection procedure. Suggestedreagent amounts for optimization are provided in the reverse transfec-tion procedure (section II.B starting on page 7), and a step-by-stepworkflow for transfection optimization is shown in section III.C start-ing on page 14.

Problems with transfection

complex formation

Follow the instructions for transfection complex formation closely;using the appropriate incubation times is important for good transfec-tion efficiency.


IV.C. Transfection Causes Extensive Cell Death18

Serum, polyanions, or other inhibitors were present during

complexing.

Although siPORT NeoFX transfection agent is compatible with serumduring transfection, it is not compatible with serum during complex for-mation. Use OPTI-MEM I reduced serum medium for Pre-miR miRNAPrecursor/siPORT NeoFX transfection agent complex formation.

Do not overmix.

It is important to gently mix Pre-miR miRNA Precursor with dilutedsiPORT NeoFX in step II.B.3.d on page 9 of the reverse transfectionprocedure, or V.A.2.d on page 20 of the traditional procedure.

Inactivated transfection

agent

Store siPORT NeoFX at 4°C and do not allow it to freeze. Also, tightlycap the tube, because evaporation can significantly impact the activity oftransfection agents.

Pre-miR hsa-miR-1 miRNA

Precursor is degraded due to

poor handling or storage

Check the integrity of the Pre-miR hsa-miR-1 miRNA Precursor byrunning ~30 μL on a non-denaturing 15–20% acrylamide gel. Visualizethe miRNA by staining with ethidium bromide, and verify that it is theexpected size and intensity. The miRNA should migrate as a fairly tightband; smearing would indicate degradation.

Cells have been subcultured

too many times or have

undergone changes

Transfect cells within 10 passages of optimization experiments.

C. Transfection Causes Extensive Cell Death

Too much transfection agent

was used

Titrate siPORT NeoFX transfection agent over a broad dilution range,and choose the most dilute concentration that still provides goodPre-miR miRNA Precursor activity.

Cells were exposed to

transfection complexes for

too long

Sensitive cells may begin to die from exposure to the transfection agentafter a few hours. If transfection causes excessive cell death with yourcells, remove the transfection mixture, and replenish with fresh growthmedium after 8–24 hr.

Cells are stressed • Add fresh growth medium as early as 8 hr after transfection.• Avoid using antibiotics when plating cells for transfection, and for at

least 48 hr after transfection. • Use healthy cells that have not been grown to the point of medium

depletion between subculturing events. • Avoid subjecting cells to frequent temperature and pH shifts.

The target gene is critical for

cell survival

If the target gene is critical for cell survival, reducing its expression couldcause cell death. If this is the case, assay for Pre-miR miRNA Precursoractivity at earlier time points (4–24 hr).

IV.D. Experiments Lack Reproducibility

Troubleshooting

19

D. Experiments Lack Reproducibility

Transfection complexes

were not adequately mixed

with cells

Distribute transfection complexes by gently rocking the plate back andforth. Do not swirl plates to mix, because this could concentrate cellsand/or reagents in the center of the wells.

There were differences in

the experimental procedure

The time of transfection after cell plating, incubation times, master mixvolumes, and the order of component addition can all affect transfectionefficiency. To obtain reproducible results in experiments involvingtransfection, conduct experiments exactly the same way every time.

Cell density is too low Optimize cell density as described in section III.C.5 on page 16. Whencell density is too low, cell cultures can become unstable. This instabilitycan vary from well to well because conditions (pH, temperature, etc.)may not be uniform across a multi-well plate, and can differentiallyinfluence unstable cultures.

Cells were passaged too

many times

Repeat experiment using cells that have been subcultured fewer times.


V.A. Traditional “Pre-plating” Transfection Procedure20

V. Appendix

A. Traditional “Pre-plating” Transfection Procedure

The following procedure is a traditional “pre-plating” method. We rec-ommend it as the first transfection method to try with AmbionsiPORT™ Amine Transfection Agent. It requires more time than reversetransfection, but may be more effective with some cell types.

IMPORTANT

The volumes and amounts in the following procedure are for transfection in

a 24 well plate.

1. Plate cells 24 hr before

transfection

a. Approximately 24 hr before transfection, plate cells in normal growthmedium (e.g., DMEM, 10% FBS) so that they will be 30–80%confluent after 24 hr.

b. Incubate the cells overnight under normal cell culture conditions.

2. Prepare Pre-miR miRNA

Precursor/transfection

agent complexes

a. Just before using it, briefly vortex the siPORT NeoFX.

b. Dilute the transfection agent into Opti-MEM I medium.

i. In a sterile, round-bottom (or V-bottom) 96 well tissue culture dish or in sterile polystyrene tube, dilute 0.5–4 μL of transfection agent dropwise into Opti-MEM I for a final volume of 25 μL.

ii. Vortex well, and then incubate at room temp 10 min.

c. Dilute 0.4–8 μL of 6.25 μM Pre-miR miRNA Precursor (for a finalconcentration of 5–100 nM) into OPTI-MEM I for a final volume of25 μL.

d. Add diluted Pre-miR miRNA Precursor to diluted transfection agent,and mix by gently flicking the tube or pipetting.

e. Incubate at room temp for 10–15 min.

Table 1. Approximate Reagent Amounts per Well for Traditional Transfection.

Procedure step Reagent 96 well 24 well 6 well

1. Plate cells 24 hr before transfection

Plate cells (per well) 0.2–1 x 104 2–10 x 104 1–5 x 105

2. Prepare Pre-miR miRNA Precursor/transfection agent complexes

Dilute transfection agent 0.15–1.2 μL 0.5–4 μL 3–9 μL

in Opti-MEM I to: to 10 μL to 25 μL to 100 μL

Dilute Pre-miR™ Precursor (6.25 μM)

0.08–1.6 μL 0.4–8 μL 2–40 μL

in Opti-MEM I to: to 10 μL to 25 μL to 100 μL

3. Transfect cells Adjust medium in wells to: 80 μL 450 μL 2300 μL

Final transfection volume ~100 μL ~500 μL ~2500 μL

Add fresh medium (24 hr) 100 μL 1 mL 3 mL

V.B. References

Appendix

21

3. Transfect cells a. Adjust the volume of normal growth medium in a well containingcells to 450 μL.

b. Add the transfection agent/miRNA complex from step 2.e dropwiseto the cells (the final transfection volume will be 500 μL).

c. Without swirling, gently rock the dish back and forth to evenlydistribute the complexes.

d. Incubate cells under normal cell culture conditions for 48 hr.

e. 0.5–1 mL fresh normal growth medium may be added to each wellafter 8–48 hr to maximize cell growth and prevent potentialcytotoxicity.

4. Assay for activity after

transfection

For initial experiments, we recommend analyzing Pre-miR miRNA Pre-cursor activity ~48 hr after transfection. Assess both PTK9 downregulationand cell viability (see sections III.A on page 11 and III.B on page 13).

B. References

Bartel D (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281–297.

Carrington J, and Ambros V (2003) Role of microRNAs in plant and animal development. Science 301: 336–338.

Lim L, Lau N, Garrett-Engele P, Grimson A, Schelter J, Castle J, Bartel D, Linsley P, Johnson J (2005) Microarrayanalysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433: 769–773.

Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAsmediate RNA interference in cultured mammalian cells. Nature 41: 494–498.

Freshney RI (2000) Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, New York(NY):Wiley-Liss.

Schramke V, Sheedy D, Denli AM, Bonila C, Ekwall K, Hannon G, Allshire R (2005) RNA-interfer-ence-directed chromatin modification coupled to RNA polymerase II transcription. Nature435(7046):1275–1279.

Yekta S, Shih I, Bartel D (2004) MicroRNA-directed cleavage of HOXB8 mRNA. Science304(5670):594–596.

C. Quality Control

Functional testing Cells were transfected with Pre-miR™ hsa-miR-1 miRNA Precursor andPre-miR Negative Control #1 using siPORT™ NeoFX™ TransfectionAgent. Cells transfected with the Pre-miR hsa-miR-1 miRNA Precursorare shown to have a 70% reduction in PTK9 mRNA levels compared tocells transfected with the Pre-miR Negative Control #1.

RNase activity testing Samples of Pre-miR hsa-miR-1 miRNA Precursor and Pre-miR Nega-tive Control #1 are incubated with labeled RNA, followed by PAGEanalysis.


V.D. Safety Information22

D. Safety Information

Chemical safety guidelines To minimize the hazards of chemicals:• Read and understand the Material Safety Data Sheets (MSDS) pro-

vided by the chemical manufacturer before you store, handle, orwork with any chemicals or hazardous materials.

• Minimize contact with chemicals. Wear appropriate personal protec-tive equipment when handling chemicals (for example, safety glasses,gloves, or protective clothing). For additional safety guidelines, con-sult the MSDS.

• Minimize the inhalation of chemicals. Do not leave chemical con-tainers open. Use only with adequate ventilation (for example, fumehood). 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 onthe MSDS.

• Comply with all local, state/provincial, or national laws and regula-tions 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 chem-ical to a customer after an MSDS has been updated. MSDSs provide thesafety information you need to store, handle, transport, and dispose ofthe chemicals safely.

Each time you receive a new MSDS packaged with a hazardous chemi-cal, be sure to replace the appropriate MSDS in your files.

Obtaining the MSDS To obtain Material Safety Data Sheets (MSDSs) for any chemical prod-uct supplied by Applied Biosystems or Ambion:• At www.appliedbiosystems.com, select Support, then MSDS.

Search by chemical name, product name, product part number, orMSDS part number. Right-click to print or download the MSDS ofinterest.

• At www.ambion.com, go to the web catalog page for the product ofinterest. Click MSDS, then right-click to print or download.

• E-mail ([email protected]) or tele-phone (650-554-2756; USA) your request, specifying the catalog orpart number(s) and the name of the product(s). We will e-mail theassociated MSDSs unless you request fax or postal delivery. Requestsfor postal delivery require 1–2 weeks for processing.

For the MSDSs of chemicals not distributed by Applied Biosystems orAmbion, contact the chemical manufacturer.

http://www.appliedbiosystems.com

Documents

Pre-miR™ miRNA Precursor Starter Kit Protocol (PN 4381862C)