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TaqMan Low Density Array: MicroRNA Profiling for Biomarker and Oncosuppressor Discovery

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Part of the Methods in Molecular Biology book series (MIMB, volume 1509)

Abstract

MicroRNAs (miRNAs) have gained a lot of interest as biomarkers and biotherapeutics in recent years. The discovery of miRNAs in circulation as recently as 2008, aided by rapid advances in high-throughput profiling techniques initiated an explosion of investigations dedicated to discovering circulating miRNAs as minimally invasive biomarkers. As miRNAs regulate many cellular processes, investigators are actively exploring their relevance in disease treatment by miRNA restoration. This chapter demonstrates an approach to discover miRNAs of biomarker and therapeutic potential by isolating miRNAs from cell lines, performing global miRNA profiling, investigating miRNA expression in clinical specimens and examining their therapeutic relevance by restoring miRNA expression using Lipofectamine in vitro.

Key words

microRNA Biomarker TaqMan low density array miRNA mimic Transfection RNAi Clinical In vitro 
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1 Introduction

miRNAs are small (18–25 nucleotides) noncoding RNAs that function intracellularly to halt messenger RNA (mRNA) translation [1, 2] subsequently regulating a plethora of cellular processes and phenotypes associated with development and disease progression [3, 4, 5]. The discovery of circulating miRNAs in 2008 stimulated a new field of biomarker research [6]. miRNAs have since been observed to be circulating in blood encapsulated within exosomes [7], microvesicles [8], part of lipoprotein–miRNA complexes [9] or as free unbound miRNA [10]. They are attractive entities for the use as biomarkers as they can be obtained minimally invasively, are stable in circulation and storage, resistant to RNAses, can be detected sensitively and specifically (by qPCR), show disease-specific expression, are evolutionarily conserved, and represent epigenetic alterations reflecting microenvironmental and cellular changes prior to and throughout disease progression [6, 7, 8].

There remain challenges in discovering effective circulating miRNAs as biomarkers. Of paramount importance to developing a reproducible, specific and sensitive biomarker is the absolute observance of the established standard operating procedure (SOP) developed by the Early Detection Research Network, as will be presented in this chapter [11]. Variables such as processing time, temperature, sample hemolysis, additives in blood collection tubes, sample storage, and excessive freeze–thaw cycles can interfere with the discovery of effective biomarkers [12]. Extra care must be taken to standardize preanalytical variables as miRNA expressions are highly sensitive to environmental alterations, including, but not limited to, exercise [13], eating patterns [14], smoking [15], and sleeping patterns [16]. Lack of following standardized protocols has, at least partly, led to difficulties in producing rugged data in miRNA biomarker research [17].

An on-going challenge in the field of miRNA biomarker research is the lack of consensus on the most appropriate endogenous control to normalize relative miRNA expressions [12]. Four options are available to researchers for miRNA expression normalization: (a): normalization to housekeeping miRNAs, however there is a lack of consensus of their accuracy due to apparent disease specific expression patters [18] (b): Global mean normalization: however, this is only statistically accurate for large datasets [19] (c): Direct CT versus CT normalization: this requires extremely precise measurements and can be used to determine relative miRNA expression per mL of serum and (d): Spike-in reference miRNA: 1.6 × 108 copies of Cel—miR-39 are spiked-in to the serum specimen during RNA isolation [12]. Here, we recommend using the spiked-in approach as it allows the researcher to normalize the data in three ways: (a): relative miRNA level per volume of starting serum, (b): quantification of miRNA copy number and (c): relative miRNA expression compared to the spiked-in control [12].

miRNA expression is typically and vastly downregulated in cancerous tumors, suggesting that loss of miRNA expression is partly responsible for tumor progression by allowing increased oncoprotein translation [20]. As miRNAs can act as potent oncosuppressors, this leaves the possibility for the use of miRNAs therapeutically as a miRNA restoration approach to cancer treatment [21]. This field of research is showing much promise, with the first miRNA restoration therapy entering a phase 1 clinical trial in 2013 for treatment of solid tumors and hematological malignancies and is well tolerated [22].

In this chapter, we present global miRNA profiling as a method to identify downregulated miRNAs, which may have therapeutic potential in control versus experimental samples. We recommend restoring miRNA expression using miRNA mimics with Lipofectamine®, a liposome forming reagent, to investigate their therapeutic potential in vitro.

To discover miRNAs of biomarker and therapeutic potential one can employ the use of global miRNA profiling by TaqMan Low Density Array (TLDA). TLDA is a quantitative reverse transcription PCR (RT-qPCR) based profiling method using 384-well micro-fluidic cards, where each well represents a miRNA of interest. The system offers a user friendly, straightforward workflow for the profiling of hundreds of miRNAs, which come either as standard or customizable cards. The TLDA procedure begins with reverse transcription (RT) of total RNA (350–1000 ng) using MegaPlex miRNA RT primers. The system also allows for the use of as little as 1 ng of RNA if performed with a pre-amplification PCR step. The RT product is then combined with TaqMan universal master mix, loaded onto the micro-fluidic cards and global miRNA profiling is performed. To identify miRNAs of biomarker/therapeutic potential the CT values are normalized to the endogenous controls (included on the cards) and relative miRNA expressions are calculated using the ΔΔCT method as will be described.

Here we outline a complete protocol detailing a translational approach to miRNA biomarker discovery, developing on in vitro profiling to clinical samples. We also present standardized protocols for serum collection to aid in the discovery of rugged miRNA based biomarkers. This method is intended to collate SOPs and best methods determined from extensive investigations to discover robust and rugged miRNA biomarkers. In addition, we also present an approach to discover oncosuppressive miRNAs by global miRNA profiling and how these miRNAs may be exploited in preclinical investigations in vitro as miRNA restoration therapies.

2 Materials

2.1 Collection of Cell Pellets

  1. 1.

    ~70 % confluent cells (control and experimental cell lines, e.g., Hs578T and Hs578Ts(i)8 triple-negative breast cancer isogenic variants).

     
  2. 2.

    Phosphate buffered saline (PBS).

     
  3. 3.

    Trypsin.

     
  4. 4.

    15 mL polypropylene centrifuge tubes.

     
  5. 5.

    1.5 mL Eppendorfs-RNase free.

     
  6. 6.

    Microcentrifuge.

     

2.2 Isolation of Total RNA (Including miRNA) from Cells

  1. 1.

    RNAseZap.

     
  2. 2.

    miRNeasy mini kit (Qiagen). Contents: RNeasy® Mini Spin columns, 1.5 mL collection tubes, 2.0 collection tubes, QIAzol® lysis regent, Bufer RWT, Buffer RPE, RNase-free water.

     
  3. 3.

    Chloroform—Harmful and should only be used in a fume hood.

     
  4. 4.

    Vortex.

     
  5. 5.

    100 % molecular grade ethanol.

     
  6. 6.

    Sterile, RNase-free pipette tips.

     
  7. 7.

    Microcentrifuge.

     
  8. 8.

    1.5 mL or 2 mL microcentrifuge tubes.

     

2.3 TaqMan Low Density Array

  1. 1.

    microRNA reverse transcription kit (for this study we used from Applied Biosystems). Contents: 100 mM dNTPs, MultiScribe reverse transcriptase 50 U/μL, 10× RT buffer and RNase inhibitor 20 U/μL.

     
  2. 2.

    MegaPlex™ RT primers (10×).

     
  3. 3.

    1.5 mL eppendorfs—RNase free.

     
  4. 4.

    96-well MicroAmp® optical reaction plate.

     
  5. 5.

    MicroAmp® clear adhesive film.

     
  6. 6.

    Thermocycler.

     
  7. 7.

    TaqMan miRNA array cards (Applied Biosystems).

     
  8. 8.

    TaqMan Universal PCR master mix, no AmpErase UNG (Applied Biosystems).

     
  9. 9.

    Nuclease-free water.

     
  10. 10.

    Sorvall or Heraeus centrifuge.

     
  11. 11.

    TaqMan® Array Micro Fluidic Card Sealer.

     
  12. 12.

    ViiA™ 7 Real Time PCR System.

     

2.4 Obtaining and Biobanking Serum Specimens

  1. 1.

    Red top Vacutainer.

     
  2. 2.

    Swinging bucket centrifuge.

     
  3. 3.

    Sterile cryovials.

     
  4. 4.

    RNase-free, filter tips.

     

2.5 Isolation of Total RNA (Including miRNA) from Serum

  1. 1.

    Serum/plasma miRNA isolation kit (we used miRNeasy Kit, Qiagen). Contents: RNeasy® MinElute® Spin columns, 1.5 mL collection tubes, 2 mL collection tubes, QIAzol® lysis regent, Buffer RWT, Buffer RPE, Ce-miR-39_1 miScript® primer assay, RNase-free water.

     
  2. 2.

    miRNeasy Serum /Plasma Spike-in Control (Qiagen).

     
  3. 3.

    Chloroform-Harmful and should only be used in a fume hood.

     
  4. 4.

    100 % molecular grade ethanol.

     
  5. 5.

    Nuclease-free water.

     
  6. 6.

    Sterile, RNase-free pipette tips.

     
  7. 7.

    Microcentrifuge.

     
  8. 8.

    RNAseZap.

     
  9. 9.

    1.5 mL or 2 mL microcentrifuge tubes.

     

2.6 Preparation of miRNA cDNA from Serum RNA

  1. 1.

    TaqMan microRNA Reverse transcription kit (Applied Biosystems). Contents: 100 mM dNTPs, MultiScribe reverse transcriptase 50 U/μL, 10× reverse transcription buffer, RNase inhibitor 20 U/μL.

     
  2. 2.

    RNase-free water.

     
  3. 3.

    1.5 mL eppendorfs-RNase free.

     
  4. 4.

    TaqMan microRNA assay (Applied Biosystems).

     
  5. 5.

    TaqMan microRNA assay kit for Cel-miR-39.

     
  6. 6.

    MicroAmp® optical reaction plate 96-well (Applied Biosystems).

     
  7. 7.

    MicroAmp® clear adhesive film (Applied Biosystems).

     
  8. 8.

    TaqMan Universal PCR master mix, no AmpErase UNG (Applied Biosystems).

     
  9. 9.

    MicroAmp® fast optical 96-well reaction plate with barcode 0.1 mL (Applied Biosystems).

     
  10. 10.

    MicroAmp® optical adhesive film (Applied Biosystems).

     
  11. 11.

    Real time PCR system (e.g., ViiA™7 Real Time PCR System).

     

2.7 Transfection of Cell Lines with miRNA Mimics

  1. 1.

    Lipofectamine® 2000 reagent (ThermoFischer).

     
  2. 2.

    mirVana™ miRNA mimic and negative control mimics (Applied Biosystems).

     
  3. 3.

    6-well tissue culture plates.

     
  4. 4.

    Opti-MEM medium.

     

3 Methods

3.1 Collection of Cell Pellets

  1. 1.

    24 h before collection, feed cells with their recommended volume of complete media.

     
  2. 2.

    Collect cell pellets from 70 % confluent, healthy cells. Do not use more than 1 × 107 cells.

     
  3. 3.

    Aspirate the medium from the cells and wash cell layer with 5 mL of PBS twice.

     
  4. 4.

    Aspirate PBS and add 5 mL of trypsin for 5 min.

     
  5. 5.

    Neutralize trypsin with 5 mL of media containing FBS.

     
  6. 6.

    Centrifuge the cell suspension at 300 × g for 5 min.

     
  7. 7.

    Discard supernatant and resuspend the cell pellet in 1 mL of PBS.

     
  8. 8.

    Centrifuge at 300 × g for 5 min. Aspirate PBS and resuspend in 1 mL of PBS.

     
  9. 9.

    Transfer to a 1.5 mL RNase-free eppendorf and centrifuge for 5 min at 13,000 × g and 4 °C using a microcentrifuge. Remove all traces of PBS and use immediately or store at −80 °C until required.

     

3.2 Isolation of Total RNA (Including miRNA) from Cells

  1. 1.

    Clean all surfaces and equipment with RNAseZap®.

     
  2. 2.

    Dilute buffer RWT and RPE with 100 % ethanol, as indicated on their bottles, to obtain a working solution.

     
  3. 3.

    Cool microcentrifuge to 4 °C.

     
  4. 4.

    Add 700 μL of QIAzol® Lysis Reagent to the cell pellet and vortex for 1 min. Allow to stand at 15–25 °C for 5 min.

     
  5. 5.

    Add 140 μL of chloroform and vortex for 15 s. Allow to stand at 15–25 °C for 3 min.

     
  6. 6.

    Centrifuge 12,000 × g for 15 min at 4 °C.

     
  7. 7.

    Warm the microcentrifuge to 22 °C.

     
  8. 8.

    Post centrifugation, three phases have developed (see Note 1 ). Carefully transfer the upper aqueous phase to a new collection tube and record the transferred volume. Ensure not to disrupt the interphase as this will result in reduced RNA purity. Discard interphase and lower organic phase.

     
  9. 9.

    Add 1.5 volumes of 100 % molecular grade ethanol to the aqueous phase and mix by pipetting up and down.

     
  10. 10.

    Immediately add 700 μL of the sample to a spin column and centrifuge at 8000 × g for 15 s at 22 °C. Discard the flow through.

     
  11. 11.

    Add the remaining solution to the spin column. Repeat centrifugation at 8000 × g for 15 s at 22 °C.

     
  12. 12.

    Add 700 μL of buffer RWT to the spin column, close the lid and centrifuge at 8000 × g for 15 s at 22 °C. Discard flow through.

     
  13. 13.

    Add 500 μL of buffer RPE to the spin column, close the lid and centrifuge at 8000 × g for 15 s at 22 °C. Discard flow through.

     
  14. 14.

    Add another 500 μL of buffer RPE to the spin column, close the lid and centrifuge at 8000 × g for 2 min at 22 °C. Discard flow through.

     
  15. 15.

    To dry the spin column membrane, place the spin column in a new 2 mL collection tube, open the lid of the spin column and centrifuge at 13,000 × g for 1 min at 22 °C.

     
  16. 16.

    Finally, to elute the captured RNA, place the spin column in a new 1.5 mL collection tube and add 50 μL of RNase-free water carefully to the center of the membrane. Centrifuge for 1 min at 13,000 × g to collect the RNA.

     
  17. 17.

    The eluted RNA can be assessed spectrophotometrically using NanoDrop to determine RNA yield and purity. A pure RNA sample is indicated by a 260/280 ratio of 2.0 (see Note 2 ).

     

3.3 TaqMan Low Density Array

  1. 1.

    Prepare RNA dilutions in 3 μL of RNase-free water per reaction so that it contains 350–1000 ng of RNA (116.67 ng/μL–333.33 ng/μL).

     
  2. 2.
    Prepare the RT master mix according to Table 1.
    Table 1

    Reverse transcription master mix

    Component

    Volume per reaction (μL)

    Volume per ten reactions (Includes 12.5 % excess) (μL)

    MegaPlex RT primers (10×)

    0.80

    9.00

    dNTPs with dTTP (100 mM)

    0.20

    2.25

    MultiScribe reverse transcriptase (50 U/μL)

    1.50

    16.88

    Reverse transcriptase buffer (10×)

    0.80

    9.00

    MgCl2 (25 mM)

    0.90

    10.13

    RNase inhibitor (20 U/μL)

    0.10

    1.13

    Nuclease-free water

    0.20

    2.25

     
  3. 3.

    Gently mix the samples and briefly centrifuge.

     
  4. 4.

    Pipette 4.5 μL of RT master mix as appropriate to each well of a 96-well MicroAmp® Optical Reaction Plate.

     
  5. 5.

    Add 3 μL of diluted RNA sample to each well as appropriate.

     
  6. 6.

    Seal the plate using MicroAmp® clear adhesive film.

     
  7. 7.

    Briefly centrifuge the plate and place on ice for 5 min.

     
  8. 8.
    Run the RT-PCR under the conditions in Table 2.
    Table 2

    RT-qPCR program

    40 cycles

    2 min

    16 °C

    40 cycles

    Hold

    1 min

    42 °C

    1 s

    50 °C

    5 min

    85 °C

    Hold

    ∞

    4 °C

     
  9. 9.

    Store cDNA at −20 °C until needed.

     
  10. 10.

    Allow the TaqMan microRNA array card to reach room temperature.

     
  11. 11.

    Thaw the RT product on ice, invert gently and briefly centrifuge.

     
  12. 12.
    Prepare the PCR reaction according to Table 3.
    Table 3

    Master mix for TaqMan low density array

    Component

    Volume for one array card (μL)

    TaqMan® Universal PCR Master Mix, No AmpErase® UNG, 2×

    450

    RT product (cDNA)

      6

    Nuclease-free water

    444

    Total

    900

     
  13. 13.

    Gently mix the samples and briefly centrifuge using a Sorvall or Hereaus centrifuge.

     
  14. 14.

    Add 100 μL of the PCR reaction mix into each port of the TaqMan array card.

     
  15. 15.

    Centrifuge the array card at 331 × g for 1 min twice using a Sorvall or Hereaus centrifuge.

     
  16. 16.

    Seal the micro fluidic cards using the TaqMan® micro fluidic card sealer

     
  17. 17.

    Load onto the ViiA™ 7 Real-Time PCR System and run using the predefined TLDA thermal cycling conditions for your TaqMan MicroRNA Array of use.

     
  18. 18.

    Analyze the CT values to determine relative miRNA expressions (see Notes 3 and 4 ).

     

3.4 Obtaining and Biobanking Serum Specimens

  1. 1.

    Blood specimens should sit at room temperature for a minimum of 30 min and a maximum of 60 min to allow the clot to form.

     
  2. 2.

    Centrifuge the blood specimens at 1200 × g for 20 min at room temperature.

     
  3. 3.

    Aliquot the serum in 250 μL volumes using RNase-free pipette. Pipette to labeled cryovials and secure cap tightly (see Note 5 ).

     
  4. 4.

    Ensure no hemolysis has occurred as would be indicated by a pink/red hue in the serum. Hemolysed specimens cannot be used for RNA analysis.

     
  5. 5.

    Promptly transfer all specimens to a labeled −80 °C box and place at −80 °C.

     

3.5 Total RNA Isolation Using Qiagen miRNeasy Serum/Plasma Kit

  1. 1.

    Clean all surfaces and equipment with RNAseZap®.

     
  2. 2.

    Dilute buffer RWT and RPE with 100 % ethanol, as indicated on their bottles, to obtain a working solution.

     
  3. 3.

    Prepare a solution of 80 % ethanol using molecular grade ethanol and RNase-free water.

     
  4. 4.

    Cool microcentrifuge to 4 °C.

     
  5. 5.
    Prepare the miRNeasy Serum /Plasma spike-in control according to Table 4.
    Table 4

    Preparation of the working solution of the Cel-miR-39 spike-in control miRNA

    Solution

    Volumes

    Copies/μL

    Storage

    Stock solution

    Add 300 μL of RNase-free water to lyophilized Ce-miR-39

    2 × 1010

    Aliquot avoid freeze–thaw cycles. Store at −80 °C.

    Dilution 1

    Add 4 μL of stock to 16 μL RNase-free water

    4 × 109

    Use immediately.

    Working solution

    Add 2 μL of Dilution 1 to 48 μL RNase-free water

    1.6 × 108

    Use immediately.

     
  6. 6.

    Thaw all serum specimens on ice.

     
  7. 7.

    Using 200 μL (see Note 6 ) of serum, add 1000 μL of QIAzol® lysis reagent and mix by pipetting up and down. Allow to stand at 15–25 °C for 5 min.

     
  8. 8.

    Add 3.5 μL of the spike-in control working solution and mix thoroughly.

     
  9. 9.

    Add 200 μL of chloroform and vortex for 15 s. Allow to stand at 15–25 °C for 3 min.

     
  10. 10.

    Centrifuge at 12,000 × g for 15 min at 4 °C.

     
  11. 11.

    Warm the centrifuge to 22 °C post centrifugation to prepare for subsequent steps.

     
  12. 12.

    Carefully transfer the upper aqueous phase to a new collection tube and record the transferred volume. Ensure not to disrupt the interphase as this will result in reduced RNA purity. Discard interphase and lower organic phase (see Note 1 ).

     
  13. 13.

    Add 1.5 volumes of 100 % molecular grade ethanol to the aqueous phase and mix by pipetting up and down.

     
  14. 14.

    Immediately add 700 μL of the sample to an RNeasy MinElute spin column and centrifuge at 8000 × g for 15 s at 22 °C. Discard the flow through and add the remaining solution to the spin column. Repeat centrifugation at 8000 × g for 15 s at 22 °C.

     
  15. 15.

    Add 700 μL of buffer RWT to the spin column, close the lid, and centrifuge at 8000 × g for 15 s at 22 °C. Discard flow through.

     
  16. 16.

    Add 500 μL of buffer RPE to the spin column, close the lid, and centrifuge at 8000 × g for 15 s at 22 °C. Discard flow through.

     
  17. 17.

    Add 500 μL of 80 % ethanol to the spin column, close the lid, and centrifuge at 8000 × g for 2 min at 22 °C. Discard flow through and the collection tube.

     
  18. 18.

    Place the spin column in a new 2 mL collection tube, open the lid of the spin column, and centrifuge at 13,000 × g for 5 min at 22 °C.

     
  19. 19.

    Place the spin column in a new 1.5 mL collection tube and add 14 μL of RNase-free water carefully to the center of the membrane. Centrifuge for 1 min at 13,000 × g.

     
  20. 20.

    The yield, size distribution and quality of the RNA can be assessed using the Agilent Small RNA Analysis Kit. Using spectrophotometric systems are not recommended due to their inability to accurately quantify low concentrations of RNA obtained from serum .

     

3.6 RT-qPCR of Serum Derived miRNA

  1. 1.

    Prepare 5 μL of a 2 ng/μL dilution of RNA with RNase-free water.

     
  2. 2.

    Add the 5 μL RNA sample to the 96-well MicroAmp® Optical Reaction Plate.

     
  3. 3.
    Prepare the RT master mix according to Table 5.
    Table 5

    Reverse transcription master mix for serum miRNA expression analysis

    Component

    Volume per reaction (μL)

    Volume per ten reactions (Includes 12.5 % excess) (μL)

    100 mM dNTPs (with dTTP)

    0.15

    1.69

    Multiscribe™ reverse transcriptase, 50 U/μL

    1.00

    11.25

    10× reverse transcription buffer

    1.50

    16.88

    RNase inhibitor, 20 U/μL

    0.19

    2.14

    Nuclease-free water

    4.16

    46.80

    Total volume

    7.00

    78.75

     
  4. 4.

    Flick and briefly centrifuge the RT master mix and place on ice.

     
  5. 5.

    Add 7 μL of RT master mix to the 5 μL RNA sample. Briefly centrifuge.

     
  6. 6.

    Briefly centrifuge the TaqMan RT primer and add 3 μL of it to the RNA/RT master mixture. Briefly centrifuge.

     
  7. 7.

    Seal the plate using MicroAmp® clear adhesive film and briefly centrifuge.

     
  8. 8.
    Run the PCR under the conditions in Table 6.
    Table 6

    Reverse transcription PCR program

    Hold

    30 min

    16 °C

    Hold

    30 min

    42 °C

    Hold

    5 min

    85 °C

    Hold

    ∞

    4 °C

     
  9. 9.

    Store the samples at −20 °C until required or keep on ice if immediately performing the qPCR step.

     
  10. 10.

    Perform all qPCR assays in triplicate per miRNA and include the endogenous control/spike-in control (Cel-miR-39) on the same plate for each sample. A “no template” control should also be included on each well for each miRNA on the plate.

     
  11. 11.
    Prepare the qPCR master mix for each miRNA primer according to Table 7.
    Table 7

    Master mix for RT-qPCR of serum miRNAs

    Component

    Volume per reaction (μL)

    Volume per ten reactions (Includes 12.5 % excess) (μL)

    TaqMan universal PCR master mix II (2×), no UNG

    10.00

    112.50

    Nuclease-free water

    7.67

    86.29

    TaqMan miRNA assay primer (20×)

    1.00

    11.25

    Total volume

    18.67

    210.04

     
  12. 12.

    Flick and briefly centrifuge the RT product and place on ice.

     
  13. 13.

    Add 1.33 μL of the RT product (cDNA) to each well as appropriate.

     
  14. 14.

    Add 18.67 μL of the qPCR master mix to each well MicroAmp® fast optical 96-well reaction plate as appropriate.

     
  15. 15.
    Seal the plate with the MicroAmp® optical adhesive film and run the qPCR according to Table 8.
    Table 8

    qPCR program

    Hold

    10 min

    95 °C

    40 cycles

    15 s

    95 °C

    60 s

    60 °C

     
  16. 16.

    Analyze the data using the ΔΔCT method (see Note 3 ) normalizing CT values to the Cel-miR-39 spike-in control.

     

3.7 Transfection of Cell Lines with miRNA Mimics

  1. 1.

    Briefly centrifuge the mirVana mimics.

     
  2. 2.

    Reconstitute the 5 nmol lyophilized miRNA mimic in 50 μL of nuclease-free water to prepare a 100 μM stock solution. Store at −20 °C.

     
  3. 3.

    Seed cells in 2 mL of antibiotic-free (see Note 7 ) media in a 6-well plate to be ~60 % confluent.

     
  4. 4.

    Allow to grow for 24 h at 37 °C.

     
  5. 5.

    24 h later, prepare a 10 μM working stock of the miRNA mimic and negative control (NC) mimics with nuclease-free water for immediate use.

     
  6. 6.

    Dilute 5 μL of Lipofectamine® in 250 μL Opti-MEM per well and mix by inverting once.

     
  7. 7.

    Incubate at room temperature for 5 min.

     
  8. 8.

    Dilute 10 nM (see Note 8 ) of miRNA mimic in 250 μL Opti-MEM and mix by inverting.

     
  9. 9.

    Incubate at room temperature for 5 min. Proceed immediately to step 10.

     
  10. 10.

    Combine the Lipofectamine/Opti-MEM mix and the miRNA mimic/Opti-MEM mix.

     
  11. 11.

    Incubate at room temperature for 20 min.

     
  12. 12.

    Add the 500 μL mixture drop by drop to the cells (see Note 9 ).

     
  13. 13.

    Culture for 48–72 h at 37 °C depending on downstream applications (see Note 10 ).

     
  14. 14.

    Transfected cells can be analyzed for phenotypic alterations and protein expression analyses (see Note 11 ) for investigation of their therapeutic effects.

     

4 Notes

  1. 1.

    Three phases develop after centrifugation: (1) an upper, colorless, aqueous phase containing the RNA; (2) a mid, white interphase containing DNA, and (3) a lower, red, organic phase containing proteins. When removing the RNA aqueous phase, care must be taken to not remove any of the other phases, as this will contaminate your sample.

     
  2. 2.

    To determine RNA purity and quantity the NanoDrop system measures sample absorbance at 260 nm and 280 nm. A ratio of 2.0 indicates a pure, high-quality RNA sample. A lower 260/280 ratio indicates protein/phenol contamination.

     
  3. 3.
    Using the CT values are automatically determined by the SDS software on your ViiA™ 7 Real Time PCR System, relative quantities of miRNAs are calculated using the ΔΔCT method by normalization to one of the predefined endogenous controls on your array card (MammU6, RNU44, or RNU48) or your spiked- in control Cel-miR-39. An outline of how to calculate fold changes using the ΔΔCT method is shown in Table 9.
    Table 9

    How to calculate fold changes using the ΔΔCT method

    C T

    Cycle threshold where miRNA is detected

    ΔCT

    CT of control sample miRNA (minus) CT of control sample endogenous control

    ΔΔCT

    CT of experimental sample miRNA (minus) ΔCT of control sample

    2(−ΔΔCT)

    Relative fold change of expression

     
  4. 4.

    To identify miRNAs of clinical relevance and to strengthen the study after miRNA expression profiling, one can use Gene Expression Omnibus (GEO2R) to analyze publically available miRNA profiles of previously performed studies on tumor/serum/plasma specimens relevant to one’s work.

     
  5. 5.

    Aliquots of 250 μL per cryovial are recommended to prevent freeze–thaw cycles.

     
  6. 6.

    It is of paramount importance to ensure that the volumes of all specimens used are kept absolutely consistent when using the spike-on approach for accurate normalization.

     
  7. 7.

    Do not use antibiotics in medium during transfection as they can interfere with Lipofectamine.

     
  8. 8.

    A considerable amount of protocol optimization may be required depending on the nature of the cell line used. Some cell lines are easily transfected, while others require more optimization. We present here the recommended starting point concentrations and time-points outlined by the manufacturer. If difficulty is observed with transfection, one can alter the mimic concentration, Lipofectamine concentration and incubation time.

     
  9. 9.

    Ensure to add the Lipofectamine mixture drop-by-drop to minimize the chance of cells undergoing osmotic shock.

     
  10. 10.

    Lipofectamine may also cause some adverse toxic effects on some cell lines. If considerable toxicity observed due to the presence of Lipofectamine in culture, one can replace the media on the transfected cells after 6 h to remove the Lipofectamine without substantially affecting transfection efficiency.

     
  11. 11.

    To investigate the predicted mRNA targets of your miRNA of interest, there are free online bioinformatics tools to predict miRNA-mRNA interactions including miRWalk, miRTarBase, TargetScan, and DianaLab.

     

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.School of Pharmacy and Pharmaceutical Sciences & Trinity Biomedical Sciences InstituteTrinity CollegeDublinIreland
  2. 2.APC Ltd.DublinIreland

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