Thermo Fisher Scientific Pre-miR™ miRNA Starter Kit User manual

Brand: Thermo Fisher Scientific

Model: Pre-miR™ miRNA Starter Kit

Type: User manual

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 Description

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

C. Required Materials Not Provided with the Kit

D. Related Products Available from Applied Biosystems

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

A. Transfection: Traditional and Reverse Transfection Methods

B. Reverse Transfection Procedure

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

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

B. Evaluating the Effectiveness of the Transfection Conditions

C. Optimizing Transfection Conditions

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

A. Problems with RT and/or PCR

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

C. Transfection Causes Extensive Cell Death

D. Experiments Lack Reproducibility

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

A. Traditional “Pre-plating” Transfection Procedure

B. References

C. Quality Control

D. 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 terms

and conditions of all applicable Limited Label Licenses. For statement(s) and/or disclaimer(s) applicable to this

product, see below.

Information in this document is subject to change without notice. Applied Biosystems assumes no responsibil-

ity for any errors that may appear in this document.

Applied Biosystems disclaims all warranties with respect to this document, expressed or implied, including but

not limited to those of merchantability or fitness for a particular purpose. In no event shall Applied Biosystems

be liable, whether in contract, tort, warranty, or under any statute or on any other basis for special, incidental,

indirect, punitive, multiple or consequential damages in connection with or arising from this document,

including but not limited to the use thereof.

Literature Citation: When describing a procedure for publication using this product, please refer to it as the

Pre-miR™ miRNA Precursor Starter Kit.

Warranty and Liability: Applied Biosystems is committed to delivering superior product quality and perfor-

mance, supported by industry-leading global service and technical support teams. Warranty information for

the accompanying consumable product is available at www.ambion.com/info/warranty in “Limited Warranty

for Consumables,” which is subject to the exclusions, conditions, exceptions, and limitations set forth under

the caption “EXCLUSIONS, CONDITIONS, EXCEPTIONS, AND LIMITATIONS” in the full warranty

statement. Please contact Applied Biosystems if you have any questions about our warranties or would like

information about post-warranty support.

Notice to Purchaser:Limited License: A license to perform the patented 5' Nuclease Process for research is

obtained by the purchase of (i) both Licensed Probe and Authorized 5' Nuclease Core Kit, (ii) a Licensed 5'

Nuclease Kit, or (iii) license rights from Applied Biosystems.

The Pre-miR™ miRNA Precursor Starter Kit contains Licensed Probe. Use of this product is covered by US

patent 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 rights

under the applicable claims of US Patents, and corresponding patent claims outside the United States, which

claim 5' nuclease methods. No right under any other patent claim and no right to perform commercial services

of any kind, including without limitation reporting the results of purchaser's activities for a fee or other com-

mercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use

only. 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 Centre

Drive, 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 are

trademarks of Applied Biosystems Inc. or its subsidiaries in the US and/or certain other countries. All other

trademarks are the sole property of their respective owners.

I.A. Background and Product Description

Introduction

I. Introduction

A. Background and Product Description

The Ambion® Pre-miR™ miRNA Precursor Starter Kit is a collection of

reagents and protocols designed to provide an introduction to

microRNA (miRNA) experiments, and facilitate optimization of trans-

fection conditions for delivery of miRNA mimics. The kit includes

everything needed to demonstrate down-regulation of the

widely-expressed twinfilin-1(PTK9) mRNA by transfection of a syn-

thetic miR-1 miRNA precursor into adherent human cultured cells. A

nontargeting negative control miRNA precursor and a TaqMan®

real-time PCR primer/probe set are provided for detection of PTK9

down-regulation using real-time reverse transcription (RT)-PCR.

Background on microRNA

(miRNA)

miRNA is a class of endogenous 17–24 nt noncoding RNAs that are

important regulators of gene expression in many organisms. Like siRNAs,

miRNAs target individual mRNAs via base-pairing interactions mediated

by the RNA-induced silencing complex (RISC) pathway. In contrast to

siRNAs, many animal miRNAs appear to effect silencing by repressing

translation rather than by inducing cleavage of cognate mRNAs (Bartel

2004, Carrington 2003). Evidence is accumulating, however, that some

animal miRNAs can reduce the level of their target mRNAs (as do the

majority of plant miRNAs), so that their effects can be monitored using

methods associated with gene expression analysis such as real-time

RT-PCR and array analysis (Lim 2005, Schramke 2005, and Yekta 2004).

Pre-miR™ miRNA

Precursors

Ambion Pre-miR™ miRNA Precursors are small, chemically modified

double-stranded RNA molecules designed to mimic endogenous mature

miRNA molecules. They are similar, but not identical, to siRNAs. The

design and chemical modification of Pre-miR miRNA Precursors ensure

that the correct strand, representing the desired mature miRNA, is taken

up into the RNA-induced silencing complex (RISC)-analogous complex

responsible for miRNA activity. In contrast to miRNA expression vec-

tors, these synthetic molecules can be used in dose response studies due

to their introduction directly into the cell by transfection or electropora-

tion. Because of their small size, they are easier to transfect than plasmid

vectors and can be delivered using conditions similar to those used for

siRNAs. They enable detailed study of miRNA biological effects via

gain-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 Description

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-miR

hsa-miR-1 miRNA Precursor and a nontargeting negative control

Pre-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 interaction

in the 3' untranslated region of the PTK9 message that results in mRNA

cleavage (Lim 2005). When transfected into human and mouse cell

lines, Pre-miR hsa-miR-1 miRNA Precursor mimics endogenous miR-1

miRNA 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 nontargeting

sequence that bears no homology to the sequences of human, mouse, or

rat transcripts. Empirically it has been shown to have no effect on PTK9

mRNA 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 the

lipid-based siPORT™ NeoFX™ transfection agent, for transfection of

Pre-miR miRNA Precursors (hsa-miR-1 and Pre-miR Negative Control

#1) into adherent cultured cells. siPORT NeoFX transfection agent

facilitates small nucleic acid transfection in a broad range of cell types

with 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 reverse

transfection, cells are mixed with transfection complexes as they adhere

to a plate after trypsinization (Figure 2 on page 7). This method

bypasses several steps of the traditional “pre-plating” transfection

method, 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 they

can be reliably transfected with siPORT NeoFX transfection agent with

minimal toxicity following these instructions.

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

Introduction

Detection of Pre-miR™

hsa-miR-1 miRNA Precursor

activity

A 20X PTK9 TaqMan® Gene Expression Assay, Hs00702289_s1, which

includes PCR primers and a TaqMan probe, is included to detect reduc-

tion of human PTK9 mRNA levels using real-time RT-PCR. (See the Note

on 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 human

This kit was developed using HeLa cells primarily because they are a

readily available cell line that can be reliably transfected using

siPORT 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 mammalian

cells can be used to obtain RNA for RT-PCR analysis. We recommend

the 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

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

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

250 μL Hs00702289_s1

20X 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 Biosystems

RT-PCR materials and

equipment

We recommend using a two-step RT-PCR strategy to evaluate Pre-miR

hsa-miR-1 miRNA Precursor activity. For normalization of the

real-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 to

generate 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 PCR

Master Mix (P/N 4324018).

• We recommend any of Applied Biosystems Real-Time PCR Sys-

tems, for example the StepOne™, StepOnePlus™, 7900, 7900HT, or

7500 Real-Time PCR Systems.

TaqMan® Gene Expression Assay for an endogenous control:

To normalize gene expression data obtained with the PTK9 TaqMan

Assay, you will need to run parallel reactions to evaluate the expression

levels of an endogenous control such as 18S rRNA or cyclophilin. We

recommend 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-free

Microfuge Tubes, P/N AM12300, Non-Stick RNase-free Microfuge

Tubes, P/N AM12350)

• Barrier tips for pipettors (Ambion P/N AM12635, AM12640,

AM12645, AM12650, AM12665)

• Applied Biosystems MicroAmp™ Optical 96-Well Reaction Plates

and Optical Adhesive Films and Optical Caps

D. Related Products Available from Applied Biosystems

siPORT™

NeoFX

™

Transfection Agent

P/N AM4510, AM4511

siPORT NeoFX Transfection Agent was developed to streamline siRNA trans-

fection procedures, cutting time and increasing reproducibility. This novel

lipid-based formulation can be used to efficiently transfect adherent cells

while subculturing, without increased cytotoxicity. This reagent is compatible

with a wide range of cell lines and experimental designs, including

high-throughput applications.

siPORT™

Amine

Transfection

Agent

P/N AM4502, AM4503

siPORT Amine Transfection Agent is an easy-to-use proprietary blend of

polyamines that delivers siRNA into mammalian cells with minimal cytotoxicity.

I.D. Related Products Available from Applied Biosystems

Introduction

Pre-miR™ miRNA Precursors

P/N AM17100, AM17101,

AM17103

mirVana™ Pre-miR™ miRNA Precursors are small, chemically modified dou-

ble-stranded RNA molecules designed to mimic endogenous mature miRNA

molecules. These ready-to-use miRNA mimics can be introduced into cells

using transfection or electroporation parameters identical to those used for

siRNAs and enable detailed study of miRNA biological effects via

gain-of-function experiments. Pre-miR miRNA Precursors are available for all

miRNAs listed in the miR Base database and custom design is available.

Anti-miR™ miRNA Inhibitors

P/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 be

introduced into cells using transfection or electroporation parameters similar

to those used for siRNAs, and enable miRNA functional analysis by

down-regulation of miRNA activity.

MagMAX™-96 Total RNA

Isolation Kit

P/N AM1830

The MagMAX™-96 Total RNA Isolation Kit is a magnetic bead based total

RNA purification system designed for rapid high throughput processing of

cells in 96 well plates. High yield and high quality total RNA can be obtained

from 100 to 500,000 cultured eukaryotic cells. The kit can also be used for

total RNA isolation from small tissue samples.

RNAqueous

-96 Kit

P/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 high

yields of intact RNA without using organic solvents (such as phenol). An

optional on-the-filter DNase treatment can be performed to ensure removal

of genomic DNA for RT-PCR applications.

High Capacity cDNA Reverse

Transcription Kit

P/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 necessary

for the quantitative conversion of 0.02–2 μg of total RNA to cDNA in a

20 μL reaction.

RETROscript

Kit

P/N AM1710

First strand cDNA synthesis kit for RT-PCR. When purchased with Super-

Taq™ thermostable DNA polymerase, this kit provides reagents, controls and

protocols for reverse transcription and PCR. Both oligo(dT) and random

primers for cDNA priming are included, as is RNase inhibitor.

TaqMan

Universal PCR

Master Mix

See 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 to

enhance reaction performance across all sample types. TaqMan Universal

PCR 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 Methods

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 the

use of chemical transfection agents. Lipid-based transfection agents,

such as siPORT™ NeoFX™ Transfection Agent, facilitate transfection by

complexing into aggregates with the negatively charged nucleic acids.

These nucleic acid-transfection agent complexes are efficiently taken up

by cells, presumably by endocytosis (Figure 1).

Traditional (Pre-plated)

transfection compared to

reverse transfection

Figure 2 shows overviews of the traditional and reverse transfection

methods. In traditional transfection of adherent mammalian cells, the

cells are pre-plated 24 hr before transfection, and cultured to recover

from trypsinization, grow, and adhere to the culture plate. We recom-

mend traditional or pre-plated transfection as the first transfection

method to try with Ambion siPORT™ Amine Transfection Agent.

Reverse transfection is a time-saving, effective alternative in which cells

are transfected as they are subcultured. Compared to the traditional

pre-plating method, equivalent or improved transfection efficiency is

seen for many of the cell types tested at Ambion. In addition, the reverse

transfection process is an entire day shorter than traditional transfec-

tion. Because cells are in suspension, a larger amount of cell surface is

exposed to transfection agent/nucleic acid complexes, and this is

thought to contribute to the improved transfection efficiency. We rec-

ommend reverse transfection as the first transfection method to try with

Ambion 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

cellular

membrane

nucleus

II.B. Reverse Transfection Procedure

Pre-miR™ miRNA Precursor Transfection

Because siPORT NeoFX Transfection Agent is supplied with this kit,

the main procedure is written for reverse transfection; however, we also

include a procedure for traditional pre-plated transfection in

section 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 nmol

of Pre-miR miRNA Precursor (both the Pre-miR hsa-miR-1 miRNA

Precursor and Pre-miR Negative Control #1) for a final concentra-

tion of 6.25 μM.

Mix thoroughly and store the Pre-miR miRNA Precursor solutions

at –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 replicate

transfections 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 transfection

agent with nucleic acid

Add transfection

complex to plated cells

Dilute transfection

agent

Harvest cells

Plate cells

and grow for 24 hr

Aliquot nucleic acid

to transfect

Incubate and assay cells

Overlay with cells

Pre-plated

Transfection

Day 1

plate cells

Day 2

transfect

Day 4

assay

Reverse

Transfection

Day 1

transfect

Day 3

assay

Pre-miR™ miRNA Precursor Starter Kit

II.B. Reverse Transfection Procedure

IMPORTANT

• Nontransfected control: cells that are mock-transfected with

Opti-MEM I medium, but no transfection agent and no Pre-miR

miRNA 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 become

overcrowded or unhealthy will improve transfection and minimize

instability in cell lines from experiment to experiment. Information

on basic cell culture technique can be found in Culture of Animal

Cells: 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 medium

to 1 x105cells/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 x104 to 1.5 x105 cells/mL. To do this, first

suspend cells at 1.5 x105 cells/mL and then dilute a portion of the

suspension further to 1 x105cells/mL and 5 x104 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 to

prepare master mixes of common reagents, with ~5–10% overage, to

limit the pipetting steps needed for your experiment and minimize

variability.

Amounts per transfection

Culture Plate Type

96 well 24 well 6 well

Volume of cells

80 μL 400 μL 2.4 mL

Number of cells

recommended for first expt

(range for optimization)

8x10

(2–20 x 10

)

4x10

(2–10 x 10

)

2.4 x 10

(1–5 x 10

)

II.B. Reverse Transfection Procedure

Pre-miR™ miRNA Precursor Transfection

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 Precursor

solution 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) from

step b with the diluted Pre-miR miRNA Precursor from step c. Mix

gently 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 complexes

from step e (previous) into the wells of the culture plate following

the volume guidelines shown below. Include 3 nontransfected con-

trol wells, containing only Opti-MEM I medium (but no Pre-miR

miRNA 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

μL

μL)

1.5

(0.75–6

μL

μL)

(4.5–36

μL

μL)

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

μL

μL)

(0.4–8

μL

μL)

(2.4–48

μL

μL)

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™ miRNA

Precursor in Opti-MEM I

Incubate 10 min

at room temp

Mix

Incubate 10 min

at room temp to form

transfection complexes

Pre-miR™ miRNA Precursor Starter Kit

II.B. Reverse Transfection Procedure

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 them

into wells of the culture plate containing Pre-miR miRNA Precur-

sor/siPORT NeoFX Transfection Agent complexes or into wells set

up as nontransfected controls.

b. Gently rock the transfection mixture.

Rock the plate gently back and forth to evenly distribute the cells and

the transfection complexes. Avoid swirling, as this can cause contents

to 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 normal

growth 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 apoptosis

before 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

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-1

miRNA Precursor is expected to reduce the amount of PTK9 mRNA in

the cells. The Pre-miR miRNA Precursor Starter Kit is supplied with a

PTK9 TaqMan® Gene Expression Assay for using real-time RT-PCR to

measure the amount of PTK9 mRNA in human cells that have been

transfected 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 with

each 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 RNA

Isolation Kit (P/N AM1830) or RNAqueous®-96 Kit (P/N AM1920) for

their 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 produce

cDNA. We recommend using either the Ambion RETROscript® Kit

(P/N AM1710) or Applied Biosystems High Capacity cDNA Reverse

Transcription Kit (P/N 4368814, 4374966) for these reactions, but any

random-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 control

for amplification of genomic DNA (without reverse transcriptase,

the sample will not contain cDNA).

• Prepare master mixes of common reagents, with ~5–10% overage to

to 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 Recommendations

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 other

words, amplify samples from each RT using both a PTK9 TaqMan

Assay 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/probe

used. This negative control helps to identify DNA contamination of

the PCR reagents.

a. Program the real-time PCR instrument.

Following are PCR cycling conditions and a suggested reaction

set-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 AmpErase

UNG requires an initial 2 min incubation at 50°C.)

b. Assemble the PCRs

Prepare a cocktail of common reagents, with ~5–10% overage to

minimize variability.

• TaqMan Gene Expression Assays are a ready-to-use 20X mixture

of forward and reverse PCR primers and a TaqMan probe. Add

0.5 μL TaqMan Assay to the PCR for a final concentration of 1X.

• We generally include 2 μL of the RT reaction (cDNA) in each

PCR. For amplification of very highly expressed endogenous con-

trol messages such as 18S rRNA, however, you may have to dilute

a portion of the RT 1:30, and use 2 μL of the diluted cDNA to

get 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 240 95°C 15 sec

60°C 1 min

Amount Component

5μL TaqMan

Universal PCR Master Mix, No AmpErase

UNG (2X)

0.5 μL TaqMan

Gene Expression Assay (20X)

2μL 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

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 in

these experiments, is inversely proportional to the quantity of the target

mRNA after normalization to the CT value for an endogenous control

gene mRNA. This normalized CT value is known as ΔCT, and for the

PTK9 amplicon, it is defined as follows:

= C

for PTK9 – C

for endogenous control

2. Calculate the percent

downregulation of PTK9

PTK9 downregulation is related to the difference in the ΔCT value for

samples transfected with Pre-miR hsa-miR-1 miRNA Precursor (ΔCT

miR-1) compared to the ΔCT value for samples transfected with

Pre-miR Negative Control #1 (ΔCTNeg#1). This is known as the ΔΔCt:

ΔΔ

miR-1 –

Neg#1

The percent downregulation can then be determined using the relation:

% downregulation = 100 – 100 X 2

–

ΔΔ

3. Evaluate transfection-

associated cytotoxicity

from CT values

Transfection agents are somewhat toxic to cells. Since transfection with

the Pre-miR Negative Control #1 miRNA Precursor will not affect gene

expression, any decrease in the expression level of the endogenous con-

trol in cultures transfected with the Pre-miR Negative Control #1 can be

attributed to cytotoxicity. Specifically, compare the CT of the endoge-

nous control amplification in cells transfected with Pre-miR Negative

Control #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 would

result 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:

Neg #1 ÷ C

nontransfected = cytotoxicity factor

4. Calculating the best

transfection condition

tested

Optimal conditions for Pre-miR miRNA Precursor transfection for a

given cell type are those which simultaneously maximize the percent

downregulation of PTK9 and minimize transfection-associated cytotox-

icity. A useful way to describe the balance of transfection efficiency and

cytotoxicity is the term Optimal Balance Factor, or OBF. The OBF is

calculated for each transfection condition as follows:

OBF = (cytotoxicity factor) X (% downregulation)

OBF = (C

Neg #1 ÷ C

nontransfected) X (100 – 100 X 2

–

ΔΔ

)

Typically, optimal transfection condition(s) are those which exhibit

highest OBF value.

Pre-miR™ miRNA Precursor Starter Kit

III.C. Optimizing Transfection Conditions

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 by

real-time RT-PCR, with ≤25% cytotoxicity or cell death. Figure 3 on

page 14 shows an overview of the transfection optimization strategy

described 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 that

will provide good Pre-miR miRNA Precursor activity while minimizing

transfection-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 miRNA

Precursor and cytotoxicity must be considered when interpreting opti-

mization experiments—with a balance between the two representing

the ideal conditions for transfection. Once optimal conditions are estab-

lished, they should be kept constant among experiments for a given cell

type.

1. Choice of transfection

agent

Ambion’s siPORT NeoFX Transfection Agent is highly effective with

minimal cytotoxic effects. It is useful for a wide variety of adherent cul-

tured mammalian cells. However, different cell types may vary in their

response to a given transfection agent. Therefore, in some instances

siPORT NeoFX may not efficiently transfect a particular cell type. In

this case, test other transfection agents such as Ambion siPORT Amine

Transfection 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 is

needed. Otherwise, optimize transfection using the strategy described here.

Optimize

transfection agent

e.g., try siPORT™ Amine

siPORT™ NeoFX™

(included in the kit)

If cytotoxicity encountered

Optimize

cell exposure to

transfection agent

To enhance overall

transfection efficiency,

optimize cell density

If no cytotoxicity

Optimize

transfection

agent volume

To optimize

down-regulation

test different

amounts of Pre-miR™

miRNA Precursor

III.C. Optimizing Transfection Conditions

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

a. For Ambion siPORT Amine Transfection Agent, follow the

procedure in section V.A. Traditional “Pre-plating” Transfection

Procedure starting on page 20. For other transfection agents, you may

follow the general procedure in section II.B. Start with a 50 nM final

concentration of Pre-miR miRNA Precursor. Follow the transfection

agent manufacturer’s recommendations for the volume of

transfection agent to use.

b. Assay for Pre-miR hsa-miR-1 miRNA Precursor activity (PTK9

down-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 to

step 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. The

overall 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 final

concentration of Pre-miR miRNA Precursor) to test four different

volumes of transfection agent (step II.B.3 of the reverse transfection

procedure), following the guidelines shown in the tables for

optimization experiments.

b. Assay for Pre-miR hsa-miR-1 miRNA Precursor activity (PTK9

down-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 minimize

cytotoxicity, exposing cells to excessive amounts of transfection agent or

for extended time periods can be detrimental to the overall health of the

cell culture.

After determining the optimal volume of transfection agent for PTK9

down-regulation, minimize cytotoxicity by adjusting the time that cells

are exposed to transfection complexes.

a. Replace the medium at 6 hr and 12 hr after transfection by carefully

aspirating the old medium from the well and adding fresh medium. It

is usually not necessary to wash cells.

Pre-miR™ miRNA Precursor Starter Kit

III.C. Optimizing Transfection Conditions

b. Re-evaluate Pre-miR hsa-miR-1 miRNA Precursor activity (PTK9

down-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 with

Pre-miR miRNA Precursor concentration. Thus, to minimize off-target

interactions, use the lowest Pre-miR miRNA Precursor concentration

which shows activity in downregulating the target gene.

To optimize the activity of transfected Pre-miR miRNA Precursors, test

5, 20, 50, and 100 nM (final concentration) Pre-miR miRNA Precur-

sor, using the transfection agent quantity and exposure time optimized

in the experiments described above.

5. Cell density For most adherent cells, the optimal confluency for transfection is

30–80%. The cell amounts listed as the range for optimization in

step II.B.2 on page 8 and Table 1 on page 20 provide guidelines for

seeding culture plates of different sizes to obtain 30–80% confluency

after 24 hr of growth; these numbers are approximate because the exact

number of cells required for seeding and transfection depends on cell

type, 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 so

that 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 in

PTK9 expression without creating instability in the cell line.

IV.A. Problems with RT and/or PCR

Troubleshooting

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 the

early cycles of PCR and the CT value is <15, it is an indication that too

much template was used in the PCR. This results in an abnormal ampli-

fication plot.

For amplification of very highly expressed endogenous control messages

such 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-time

PCR for good results.

No signal RNA contains inhibitors

If PTK9 is not detected even in samples that were transfected with the

Pre-miR Negative Control #1, it may indicate that the RNA contains

inhibitors. Samples containing minimal amounts of inhibitors may

yield successful RT-PCRs by adding less sample (and therefore less

inhibitor), to the reaction. Accomplish this by diluting samples, for

example 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 negative

control reaction is contamination of PCR reagents with DNA (often

from previous PCRs). In this case, repeat the PCR with fresh reagents

and 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. Suggested

reagent amounts for optimization are provided in the reverse transfec-

tion procedure (section II.B starting on page7), and a step-by-step

workflow 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.

Page is loading ...

Thermo Fisher Scientific Pre-miR™ miRNA Starter Kit User manual

Brand: Thermo Fisher Scientific

Model: Pre-miR™ miRNA Starter Kit

Type: User manual

Download PDF

report

Thermo Fisher Scientific Pre-miR™ miRNA Starter Kit User manual

Thermo Fisher Scientific Pre-miR™ miRNA Starter Kit User manual

Related papers

Other documents