PROTOCOL

Co-transfection of miRIDIAN™
microRNA reagents and plasmidusing DharmaFECT™ Duotransfection reagent

This protocol is optimized for use with 100 ng/well plasmid and
50 nM miRIDIAN miRNA mimic or 25 nM miRNA inhibitor in a 96-wellplate format. Plasmid concentration may need to be optimized as
plasmid size and expression attributes affect optimal conditions. The
mimic or inhibitor concentration may be titrated down once optimaldelivery conditions are established.

Additional materials required
Transfection experiments require standard cell culture reagents andinstruments appropriate for maintenance of cells. Reagents for assaying
cell viability and miRNA regulation are also needed. Table 2 lists specificreagents required, in addition to DharmaFECT Duo transfection reagent(Cat# T-2010-xx).

The samples listed in Table 1 are recommended in every transfection
experiment. All steps of the protocol should be performed in a laminarflow cell culture hood using sterile technique. Performing each sample intriplicate wells is recommended to allow statistical analysis of the results.

Table 2. Reagents for plasmid and miRNA mimic/inhibitor co-transfection experiment

Table 1. Recommended samples for plasmid and miRNA mimic/inhibitor
co-transfection experiment

Samples

PurposePlasmid only (with lipid)[Tube 1a]Confirm uptake and baseline
expression level of plasmid

Plasmid and negative control
miRNA mimic/inhibitor(with lipid)[Tube 1b]

Distinguish miRNA regulation from
non-specific effects

Plasmid and test miRNA mimic/
inhibitor (with lipid)[Tube 1c]

Achieve miRNA regulation andexpression of plasmid

Mock-transfection(lipid only)[Tube 1d]

Identify nonspecific effects andcytotoxicity caused by thetransfection reagent or procedure

Untreated (no lipid)

Determine baseline phenotype,target miRNA level, and cell viability

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Reagents

Description and use

Cells

For best transfection efficiency, use cells in log-phase growth
at a low passage number

Antibiotic-freecomplete medium

Medium in which the cells are cultured, which may contain up
to 20% serum, but does not contain antibiotics that may cause
cell toxicity during transfection

Serum-free or
low-serum medium

For optimal complexing of miRNA mimic/inhibitor, plasmidand DharmaFECT Duo transfection reagent

Plasmid

A plasmid that expresses the desired gene

Test miRNA
mimic/inhibitor

miRIDIAN microRNA Mimic/Inhibitor that targets the miRNA
to be regulated

Negative control
miRNA

A miRNA that does not target any miRNA expressed by theplasmid or any miRNA endogenously expressed by the cells
being used, such as miRIDIAN microRNA Mimic Negative
Contro (Dharmacon Cat # CN-001000-01)

Table 3. Recommended conditions for miRNA mimic/inhibitor and plasmid cotransfection using DharmaFECT Duo. The indicated number of cells was plated in
96-well plate format and incubated overnight as described in the protocol. The
psiCHECK™-2 Vector (Promega, 100 ng/well) and Renilla luciferase-targeting siRNA(100 nM) were complexed with a range of DharmaFECT Duo volumes (0.05 - 0.6 µL/well). Firefly luciferase expression was assessed 48 hours post-transfection using theDual-Glo® Luciferase Assay System (Promega). Renilla luciferase mRNA knockdown
was assessed using branched DNA analysis (Panomics, Fremont, CA). Cell viability wasassessed using the alamarBlue™ Assay (Thermo Fisher Scientific). Cell viability andsilencing values are normalized to untreated cells.

Cell line

Final cell #per wellStock celldensity(cells/mL)

VolumeVolume ofFinal volume ofof DharmaFECT serum-freeDharmaFECT
Duo (µL)
medium (µL) Duo (µL/well)

ES-D3

2.0 x 104

2.0 x 105

4.2

135.8

0.3

HeLa

2.0 x 104

2.0 x 105

1.4

138.6

0.1

Hep G22.0 x 104

2.0 x 105

0.7

139.3

0.05

Jurkat

6.0 x 104

6.0 x 105

8.4

131.6

0.6

MCF7

4

1.0 x 105

1.0 x 10

2.8

137.2

0.2

MCF10a1.0 x 104

1.0 x 105

2.8

137.2

0.2

NIH/3T3

1.0 x 104

1.0 x 105

5.6

134.4

0.4

Cell plating
Optimal cell number for plating will vary with growth characteristics ofspecific cells and may need to be determined empirically.
1. Trypsinize and count cells.
2. Dilute cells in antibiotic-free complete medium to the appropriate stock
density as described in Table 3.
3. Plate 0.1 mL cell suspension into each well of a 96-well plate.
4. Incubate cells at 37 °C with 5% CO2 overnight.
Table 4. Recommended miRNA mimic/inhibitor volumes for specific final
concentrations. This protocol is optimized for use with 100 ng/well plasmid and 50 nM
miRNA mimic or 25 nM miRNA inhibitor in a 96-well plate format. It is recommended
to start with 50 nM miRNA mimic or 25 nM miRNA inhibitor and once optimal delivery
conditions are established to titrate down to the lowest concentration of the miRNA
reagent that still produces acceptable miRNA regulation. *Recommended initial volumes
Final mimic concentration (nM)

Volume of 2 µM mimic Final inhibitor
Volume of 2 µM inhibitor
stock solution (µL)
concentration (nM) stock solution (µL)

50

8.5*

25*

4.2*

40

6.8

20

3.4

30

5.1

15

2.5

20

3.4

10

1.7

10

1.7

1

0.2

Transfection
All volumes are multiplied by 3.5 to miRNA mimic/inhibitor account for the
triplicate samples and loss during pipetting. This will result in 12 transfectionsin 96 well plate format: 4 combinations (tube 1a to d) in triplicate.
1. Prepare stock plasmid (20 µg/mL) and miRNA mimic/inhibitor (2 µM)
solutions in an RNase-free, pH 7.4-buffered solution.
2. In tubes 1a–1d, mix plasmid and the appropriate miRNA mimic/inhibitor
solutions, if appropriate:a. Tube 1a (plasmid only)—Dilute 17.5 µL of stock plasmid (20 μg/mL)with 17.5 µL serum-free medium. The total volume is 35 µL.
b. Tube 1b of stock plasmid (20 μg/mL) and negative control miRNA
mimic/inhibitor)—Mix 17.5 µL of stock plasmid (20 μg/mL) with
17.5 µL negative control miRNA mimic/inhibitor at 2µM. The total
volume is 35 µL.
c. Tube 1c (plasmid and test miRNA mimic/inhibitor)—Mix 17.5 µL ofstock plasmid (20 μg/mL) with the appropriate amount of test miRNA
mimic/inhibitor at 2µM (See Table 4), it is recommended to startwith 8.5 µL mimic and 4.2 µL inhibitor). The total volume is 26 µL for
mimics and 21.7 µL for inhibitors. Adjust to 35 μL with water.
d. Tube 1d (lipid only)—Do not add plasmid or miRNA mimic/inhibitor.
Adjust to 35 μL with water.
3. In tube 2, dilute sufficient DharmaFECT Duo in serum-free medium togive a total volume of 140 µL, as indicated in Table 3. This volume needs
to be increased when more than 4 tubes are considered.
4. Mix the contents of all tubes gently by pipetting carefully up and down.
5. Incubate tubes for 5 minutes at room temperature.
6. Add 35 µL of Tube 2 content to Tube 1a–1d, bringing the total volume to
70 µL. Mix by pipetting carefully up and down.
7. Incubate for 20 minutes at room temperature.
8. Add 280 µL antibiotic-free complete medium to each mix in step 6. The
total volume is 350 µL. These are the transfection media.
9. Remove medium from the wells of the 96-well plate containing cells andreplace with 100 µL of the appropriate transfection medium to each well.
10. Incubate cells at 37° C in 5% CO2 for 24–48 hours (for mRNA analysis) or
48–96 hours (for protein analysis).
11. If cell toxicity is observed after 24 hours, replace the transfection
medium with complete medium and continue incubation. Cell viability
may be determined with alamarBlue™, MTT, or other assays for metabolic
activity. For best results, use samples with at least 70% cell viability.

*Recommended initial volumes

If you have any questions, contact

psiCHECK-2 Vector and Dual-Glo are trademarks of Promega. Renilla is a trademark of Panomics. alamarBlue Assay is a trademark of Thermo Fisher
Scientific All trademarks are the property of Horizon Discovery Company unless otherwise specified. ©2018 Horizon Discovery Group Company—All
rights reserved. First published June 2014. UK Registered Head Office: Building 8100, Cambridge Research Park, Cambridge, CB25 9TL, United Kingdom.

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