Pre-miR™ miRNA Precursor Starter Kit Protocol (PN 4381862C) · Pre-miR™ miRNA Precursor Starter...
Transcript of Pre-miR™ miRNA Precursor Starter Kit Protocol (PN 4381862C) · Pre-miR™ miRNA Precursor Starter...
Pre-miR™ miRNA Precursor Starter Kit
Part Number AM1540
Cov_Pre-miR miRNA Starter.fm Page 1 Monday, August 23, 2010 2:37 PM
Pre-miR™ miRNA Precursor Starter Kit
(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
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© 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
Pre-miR™ miRNA Precursor Starter Kit
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.
Pre-miR™ miRNA Precursor Starter Kit
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.
Pre-miR™ miRNA Precursor Starter Kit
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
Pre-miR™ miRNA Precursor Starter Kit
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
Pre-miR™ miRNA Precursor Starter Kit
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.
Pre-miR™ miRNA Precursor Starter Kit
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
Monitoring Pre-miR™ hsa-miR-1 miRNA Precursor Activity
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.
Pre-miR™ miRNA Precursor Starter Kit
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
Monitoring Pre-miR™ hsa-miR-1 miRNA Precursor Activity
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.
Pre-miR™ miRNA Precursor Starter Kit
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.
Pre-miR™ miRNA Precursor Starter Kit
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.
Pre-miR™ miRNA Precursor Starter Kit
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.
Pre-miR™ miRNA Precursor Starter Kit
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.