A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics

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Molecular Therapy
Oct 2016



Primary neurons represent an ideal cellular system for the identification of therapeutic oligonucleotides for the treatment of neurodegenerative diseases. However, due to the sensitive nature of primary cells, the transfection of small interfering RNAs (siRNA) using classical methods is laborious and often shows low efficiency. Recent progress in oligonucleotide chemistry has enabled the development of stabilized and hydrophobically modified small interfering RNAs (hsiRNAs). This new class of oligonucleotide therapeutics shows extremely efficient self-delivery properties and supports potent and durable effects in vitro and in vivo. We have developed a high-throughput in vitro assay to identify and test hsiRNAs in primary neuronal cultures. To simply, rapidly, and accurately quantify the mRNA silencing of hundreds of hsiRNAs, we use the QuantiGene 2.0 quantitative gene expression assay. This high-throughput, 96-well plate-based assay can quantify mRNA levels directly from sample lysate. Here, we describe a method to prepare short-term cultures of mouse primary cortical neurons in a 96-well plate format for high-throughput testing of oligonucleotide therapeutics. This method supports the testing of hsiRNA libraries and the identification of potential therapeutics within just two weeks. We detail methodologies of our high throughput assay workflow from primary neuron preparation to data analysis. This method can help identify oligonucleotide therapeutics for treatment of various neurological diseases.

Keywords: Primary cortical neurons (原代皮质神经元), siRNA (siRNA), Screening (筛查), Branched DNA (分支DNA), QuantiGene 2.0 (QuantiGene 2.0)


Oligonucleotide therapeutics represent a new class of drug that can target any genetically defined disorder, by silencing the expression of mutant proteins. Specifically, siRNAs are double stranded oligonucleotides that are loaded into the RNA induced silencing complex (RISC) and can silence mRNA before it is translated. However, unmodified siRNAs are unstable and cannot enter cells without the help of cationic lipid formulation, which can be toxic to primary cells such as neurons. In this protocol, we use self-delivering, hydrophobically modified siRNAs (hsiRNAs) for mRNA silencing. Recent progress in the chemistry of oligonucleotides has enabled the design of these stabilized hsiRNAs, which promote cellular internalization, efficient entry into RISC, and potent knockdown of target genes (Byrne et al., 2013; Alterman et al., 2015; Ly et al., 2017). These compounds contain 2’-O-methyl and 2’-fluoro modifications on all sugars and phosphorothioate backbone modifications; the oligonucleotides are often conjugated to a hydrophobic moiety, such as cholesterol, to support membrane binding and cellular internalization without toxicity. This new class of compounds offers researchers a straightforward method for silencing various genes in the context of biologically relevant, and hard to transfect, primary cortical neurons (Alterman et al., 2015).

Today, in the early stage of drug discovery, most high-throughput tests are performed in cell-based assays for lead oligonucleotide identification and validation. Cell-based assays improve and accelerate drug screening, providing more relevant in vivo biological information than biochemical assays and reducing the need for animal testing. High-throughput assays performed in primary neurons have emerged as a powerful tool to discover new therapies for the treatment of neurodegenerative disorders, such as Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS) or Alzheimer’s disease (AD) (Sharma et al., 2012). Cell based assays using primary neurons provide a more natural (relevant) environment for studying neurodegenerative disorders than clonal neuronal lines. Transfecting oligonucleotide-based therapeutics into primary neurons generally relies on approaches such as electroporation (Mertz et al., 2002; Gresch et al., 2004; Zhang et al., 2016), viral transduction (Naldini et al., 1996; Hughes et al., 2002; Janas et al., 2006) or lipid-mediated transfection (Ohki et al., 2001; Dalby et al., 2004; Zhang et al., 2016). However, these methods can be laborious, show low efficiency, and induce cellular toxicity. Thus, the self-delivering properties of hsiRNAs represent an effective method to identify new leads in vitro in complex cellular models, such as primary neurons. We have recently demonstrated that hsiRNAs efficiently bind neuronal-cell membranes within seconds after treatment, enter cells, and induce potent gene silencing, both in vitro, in primary neurons, and in vivo, in mouse brain, all without the use of transfection reagents (Alterman et al., 2015; Ly et al., 2017).

Our laboratory has established a rapid high-throughput platform to identify and validate hsiRNA leads in primary neurons in vitro in 96-well format. To quantify the amount of target mRNA upon hsiRNA treatment, we use the QuantiGene 2.0 branched DNA (bDNA) assay, a high-throughput, 96-well plate-based mRNA quantification assay. This technique is designed to directly quantify the target mRNA from sample lysate without the need to purify the RNA, thus minimizing sample manipulation (Kern et al., 1996; Coles et al., 2015). This assay enables accurate and precise detection of even low abundance mRNAs, minimizing experimental variability and error (Collins et al., 1997; Canales et al., 2006). The combination of both self-delivering hsiRNAs and high-throughput quantification of mRNA accelerates the identification of efficacious oligonucleotides in primary neurons.

Here, we describe the workflow of this high throughput assay for performing large-scale screening and dose response validation of hsiRNAs in primary cortical neurons. We detail our methods for primary cortical neuron preparation in 96-well plate format (Figure 2), neuronal treatment with hsiRNA, assay plate management, mRNA quantification, and data analysis. This platform can be used for the screening of oligonucleotide therapeutics in primary neurons for the potential treatment of neurodegenerative diseases.

Materials and Reagents

  1. Poly-L-lysine pre-coated tissue culture treated 96-well plate (Corning, catalog number: 356516 )
  2. Deep 96-well sterile polypropylene plate (Corning, Axygen®, catalog number: 391-04-062 )
  3. Tips (from 0.2 μl to 1 ml) (VWR)
  4. Serological pipettes, individually wrapped (from 5 ml to 50 ml) (Costar)
  5. Fire-polished Pasteur pipet with cotton plug (made in-house)
  6. Tissue culture treated 10 cm dish (Corning, catalog number: 430167 )
  7. 50 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352097 )
  8. 15 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352098 )
  9. 1.7 ml microcentrifuge tubes (Genesee Scientific, catalog number: 22-282 )
  10. 1 ml sterile syringe (BD, catalog number: 309659 )
  11. Adhesive plate seals (VWR, catalog number: 60941-126 )
  12. Pregnant wild-type mice (THE JACKSON LABORATORY)
  13. Cholesterol-conjugated hsiRNAs (designed and produced in-house)
  14. Ice-cold block (Koolit® Refrigerants) (Cold Chain Technologies, catalog number: 305F )
  15. Poly-L-lysine (Sigma-Aldrich, catalog number: P4707 )
  16. Phosphate-buffered saline (PBS) (Mediatech, catalog number: 21-031-CV )
  17. 200-Proof ethanol (Decon Labs, catalog number: 2701 )
  18. DMEM cell culture medium (Mediatech, catalog number: 10-013-CV )
  19. Hibernate E (BrainBits, catalog number: HE )
  20. DNase I (Worthington, catalog number: 54M15168 )
  21. Papain (Worthington, catalog number: 54N15251 )
  22. Trypan blue stain solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  23. QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: QS0011 )
  24. QuantiGene 2.0 Probe sets (varies by gene)
  25. Lysis mixture (Thermo Fisher Scientific, AffymetrixTM, catalog number: 13228 )
  26. Proteinase K (Thermo Fisher Scientific, AffymetrixTM, catalog number: QS0103 )
  27. Neurobasal medium NbActiv4 (BrainBits, catalog number: Nb4-500 )
  28. Fetal bovine serum (FBS) (Mediatech, catalog number: 35-010-CV )
  29. NeuralQTM medium (Sigma-Aldrich, catalog number: N3100 ) supplemented with GS21TM supplement (50x) (Sigma-Aldrich, catalog number: G0800 ), 0.5 mM dipeptide Ala-Gln (Sigma-Aldrich, catalog number: A8185 )
  30. 5’UTP (Sigma-Aldrich, catalog number: U6625 )
  31. 5’FdU (Sigma-Aldrich, catalog number: F3503 )
  32. Papain/DNase solution (see Recipes)
  33. Plating medium (see Recipes)
  34. Feeding medium (see Recipes)


  1. Micropipettes from 0.5 μl to 1 ml (Labnet International, model: BioPetteTM Plus )
  2. Multichannel (8 or 12 channels) micropipettes from 10 μl to 300 μl (Eppendorf, model: Research® plus )
  3. Tissue culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i )
  4. Biological safety cabinet connected to vacuum (Thermo Fisher Scientific, Thermo ScientificTM, model: 1300 Series Class II, Type A2 )
  5. Bunsen burner
  6. Germinator 500 (Braintree Scientific, model: Germinator 500, catalog number: GER 5287-120V )
  7. Water bath at 37 °C (Fisher Scientific, model: Model 2332 )
  8. 4 °C fridge
  9. Microscissors (Fine Science Tools, catalog numbers: 14060-10 and 14002-12 )
  10. Set of two forceps (Fine Science Tools, catalog number: 11251-30 )
  11. Dissection microscope (Motic, model: SMZ168 Series )
  12. Pipet-aid (Drummond Scientific, model: Portable Pipet-Aid® XP )
  13. Tissue culture phase-contrast inverted microscope (Motic, model: AE2000 )
  14. Hemocytometer (0.1000-0.0025 mm2) (Neubauer)
  15. µPlate carrier (Beckman Coulter, model: SX4750 )
  16. Autoplate washer (BioTek Instruments, model: ELx405 )
  17. Refrigerated swing rotor benchtop centrifuge (Beckman Coulter, model: Allegra X-15R )
  18. Allegra X-15R rotor (Beckman Coulter, model: SX4750 )
  19. Plate reader spectrophotometer (Tecan Trading, model: Infinite M1000 Pro )


  1. Microsoft Office Excel (Microsoft Office)
  2. GraphPad Prism 7 software (GraphPad Software)


  1. Poly-L-lysine coating of tissue culture treated 96-well plates
    1. To coat the plates, transfer 100 μl of 0.01% poly-L-lysine to each well using a multichannel micropipette.
    2. Incubate overnight in the tissue culture incubator at 37 °C.
    3. The next day, before the dissection, aspirate the coating solution using a vacuum-connected Pasteur pipet (or a 200 μl multichannel micropipette) and rinse three times with 100 μl PBS.
    4. Remove the final wash and let the plate dry in the biological safety cabinet.
      Note: It is also possible to purchase and use 96-well plates pre-coated by the manufacturer, to store at room temperature. We did not observe any difference between data from cells plated in 96-well plates pre-coated in our laboratory or by the manufacturer.

  2. Preparation of cotton-plugged, fire-polished Pasteur pipet
    1. Using a Bunsen burner, polish the cotton plugged Pasteur pipet extremity by holding the pipet oriented with the capillary tube in the flame for 2-3 sec, rotating rapidly.
    2. Check the opening of the capillary tube, ensuring that the edge is round and the hole is not too small. If the hole is too small, discard the pipet, as it will sheer neurites during the trituration (Figure 1).
    3. Autoclave the Pasteur pipets to sterilize before use.

      Figure 1. Fire-polished pipettes. A. Cotton-plugged, fire-polished pipettes; B. From left to right: unpolished tip (edges are too sharp–pointed with white arrow), correctly polished tip (edges are smooth–pointed with green arrow), over-polished tip (opening is too small–pointed with red arrow); C. Same as in B.

  3. Isolation of mouse primary cortical neurons (Figure 2)
    Note: To avoid contamination, sterilize instruments for use during the preparation. Between each step, tools can be kept in 70% ethanol or sterilized by heat in a Germinator.
    Primary cortical neurons are isolated from E15.5 mouse embryos of pregnant mice. Before starting, prepare plating medium required during the procedure and, pre-warm at 37 °C by incubation in a water bath.

    Figure 2. Schematic of primary cortical neuron isolation

    1. Euthanize pregnant mice with CO2 followed by cervical dislocation.
      Note: This is one example of proper procedure, each investigator should follow his or her approved IACUC protocol.
    2. Lay the mouse on her back and clean the abdominal surface with 70% ethanol to sterilize the area.
    3. Using microscissors, make a vertical incision in the abdominal muscles to open the abdominal cavity of the mouse. Using forceps, carefully remove the embryonic sacs and rinse them in cell culture medium (DMEM, ice cold, no additives).
    4. Place the embryonic sacs in a 10 cm dish containing 10 ml of ice-cold DMEM.
    5. Using microscissors and forceps, carefully open the embryonic sacs, remove the embryos and transfer to a 10 cm dish containing 10 ml of ice-cold DMEM.
    Note: Steps C6 to C10 are performed one embryo after another.
    1. Place the forceps near the back of the embryos skull and pull forward toward the nose.
    2. Place the forceps on either side of the head, beneath the brain, and carefully lift the brain out of the skull (Video 1).

      Video 1. Removal of the brain from the skull

    3. Transfer the brains to a 10 cm dish containing 10 ml of ice-cold Hibernate E.
    4. Transfer brains to single droplets of Hybernate E in 10 cm dishes under the dissecting microscope for easy access.
    5. Under a dissecting microscope, use forceps to carefully peel away the meninges from the upper surface of each brain hemisphere. The cortex will fall away from the brain after the meninges are removed (Figure 3B).
    6. Using the forceps, carefully separate the cortices from the brain.

      Figure 3. Remove the meninges to release the cortices. A. Individual brain removed from skull; B. Cortex from one hemisphere following removal of the meninges; C. Cortex from one hemisphere (dotted line). Area of cortex to be transferred to papain/DNase (solid line).

    7. Transfer the upper, outer portion of the cortices (Figure 3C) into a 1.5 ml microcentrifuge tube containing 1 ml of pre-warmed papain/DNase solution and close the tube while collecting the remainder of the cortices to keep away as much contamination as possible.
    8. Incubate the tube open, in a sterile 10 cm dish for 30 min at 37 °C to dissolve the tissue. This allows the tissue to breathe while keeping it in a sterile environment.
    Note: Perform all the following steps in a biological safety cabinet to avoid any contamination.
    1. Place the tube upright for a few minutes to allow the cortices to sediment at the bottom of the tube.
    2. Without disturbing the cortices, slowly and carefully remove the papain/DNase solution using a 1 ml micropipette (be sure to remove as much papain/DNase solution as possible) and add 1 ml of plating medium.
    3. Dissociate the cells with a gentle and consistent motion of pipetting up and down several times through a cotton-plugged, fire-polished Pasteur pipet using a pipet-aid adjusted to the lowest speed. It is important to avoid the formation of bubbles. Triturate the tissue until the medium turns slightly cloudy, and no remaining tissue clumps are visible (Video 2).

      Video 2. Trituration of cortices

    4. Transfer the cells to a 15 ml conical tube and add 4 ml of plating medium. To ensure small clumps of cells are broken up, perform an additional trituration by gently and consistently pipetting up and down several times through a cotton-plugged, fire-polished Pasteur pipet using a pipet-aid adjusted to the lowest speed.
    5. Let the solution sit for 2 min to allow any debris to settle to the bottom of the 15 ml tube
    6. Transfer the supernatant containing the single cell neuronal suspension to a new 15 ml tube.
    7. Transfer a small drop of cells to a tissue culture treated dish and, using a tissue culture microscope (10x magnification), observe if there is a single cell suspension (Figure 4). If not, perform another trituration using a cotton-plugged, fire-polished Pasteur pipet, as previously described.

      Figure 4. Primary cortical neuron morphology. Phase contrast images (4x objective) of mouse primary cortical neurons showing the neuronal morphology acquisition over time.

    8. Using trypan blue stain solution and a hemocytometer, count the live cells present in the cell suspension. Add 10 μl of cells to 90 μl of trypan blue stain solution and mix gently by pipetting. Transfer a 10 μl aliquot to the hemocytometer and count the live cells from all four quadrants of the hemocytometer. Average the four numbers and multiply by 1 x 105. This number corresponds to the number of cells per ml of medium. On average, each pup will yield ~5-10 x 106 cells.
    9. Transfer the cells to a 50 ml conical tube and adjust the density of viable cells with pre-warmed plating medium to 1 x 106 cells/ml. For example, if the cell concentration is 5 x 106 cells/ml in 5 ml, add 20 ml of plating medium to obtain a final cell concentration of 1 x 106 cells/ml in 25 ml final volume.
    10. Close and invert the tube to ensure a thorough mixture of the cell suspension. Transfer the cells to a 50 ml reagent reservoir.
    11. Using a 200 μl multichannel micropipette, transfer 100 μl of cells (1 x 105 cells) to each well of a poly-L-lysine pre-coated 96-well plate. To seed an equal number of cells in each well, mix the cells in the reservoir by pipetting up and down before each transfer into the 96-well plate.
    12. Incubate the plates overnight at 37 °C, 5% CO2 in a tissue culture incubator.
    13. The following day, using a multichannel micropipette, slowly add 100 μl of feeding medium (containing anti-mitotics) to each well to prevent the growth of non-neuronal cells. Be careful not to disturb the cells.
    14. Every 3-4 days, replace half of the volume of media with freshly prepared feeding medium. Using a multichannel micropipette, slowly remove 100 μl of medium from each well without touching the bottom of the well to avoid disturbing the cells. Very gently, add back 100 μl of freshly prepared and pre-warmed feeding medium without disturbing the cells.
    15. Store in the tissue culture incubator at 37 °C. Repeat feeding step as necessary.

  4. Treatment of primary neurons with oligonucleotide therapeutics
    Note: Oligonucleotides are stored at -20 °C. Prior to any experiment, thaw the compounds on ice. This procedure is performed in a biological biosafety cabinet.
    Prepare the oligonucleotide dilutions in feeding medium. Carefully design your plate map to include the oligonucleotides of interest (hsiRNATarget), the non-targeting control (hsiRNANTC) and the untreated cells. Compound is prepared at two times the final concentration so that 50 µl compound solution can be added to 50 µl of the conditioned medium. Be sure to prepare enough compound to treat cells in the number of desired replicates. For the purposes of this protocol, we describe a preparation for three replicate wells per compound and a final concentration of 1.5 µM hsiRNA.
    1. In a deep 96-well plate, prepare a 2x hsiRNA master mix by diluting the stock hsiRNA in fresh feeding medium containing anti-mitotics to a final concentration of 3 µM (two times the final concentration of 1.5 µM) in a final volume of 200 µl.
    2. Mix gently up and down with the micropipette.
    3. Using a micropipette, gently remove the entire conditioned medium from the neurons and immediately add back 50 µl of conditioned medium to each well (you can combine the conditioned medium into a sterile reagent reservoir for easy pipetting back into the plate). Only remove medium from one or two rows at a time to prevent the cells from drying. To avoid disturbing the cells, pipet down the medium slowly with the tips touching the edge of the wells. This step is intended to control for any evaporation that may have happened during incubation at 37 °C, and to ensure that all cells are treated with the same concentration of hsiRNA.
    4. Using a multichannel micropipette and clean tips for each row, transfer 50 μl of the 2x hsiRNA master mix from the deep 96-well plate into each of the replicate wells of the cell culture plate for a final volume of 100 µl per well.
    5. Incubate the plate in a tissue culture incubator at 37 °C. For a one week long incubation, feed the cells once with 100 µl freshly prepared feeding medium (3-4 days after treatment). Then lyse the cells one week after treatment. This step ensures that no hsiRNA will be removed from the wells during the experiment. For longer term experiments, where cells need to be fed more than once, remove 100 µl of conditioned medium from the 200 µl in the cell culture plate and add back 100 µl of fresh medium. Continue this pattern of feeding every 3-4 days.

  5. mRNA quantification
    mRNA is quantified using the QuantiGene 2.0 bDNA Assay (Affymetrix). All the volumes used in the following steps are for one plate. Prepare all the solutions used for this procedure in clean 50 ml conical tubes using clean serological pipettes.
    1. In a 50 ml conical tube, prepare the diluted lysis mixture (DLM; 1:2 v/v, lysis mixture/water, e.g., 10 ml lysis mixture:20 ml water) complemented with 100 μl of 50 μg/μl Proteinase K (provided with the kit) and pour it into a 50 ml reservoir. Diluted lysis mixture should be prepared fresh for every experiment. Storing the diluted lysis mixture in the fridge will cause precipitate to form.
    2. Remove all growth medium from the plate.
    3. Using a 300 μl multichannel pipette, add 250 μl of diluted lysis mixture in each well of the 96-well culture plate. There is enough lysate to perform the quantification of the target and the control mRNA.
    4. To lyse the cells, gently pipette up and down avoiding the formation of bubbles. Use clean tips between each row.
    5. Incubate for 30 min at 55 °C in a forced air oven.
    6. Using a 300 μl multichannel pipette setup at 150 µl, mix the cell lysate thoroughly by gently pipetting up and down avoiding the formation of bubbles.
    7. Add 20 μl of probe set, diluted according to the kit protocol to each well of the capture plate (provided with the kit).
    8. Using a 100 μl multichannel pipette, transfer the appropriate amount of each lysate to the capture plate. Avoid touching the bottom of the coated plate. For background wells, simply add DLM (no Proteinase K) instead of cell lysate. Prior to testing a new gene using the QuantiGene 2.0 kit, you will need to validate the correct volume of lysate to obtain the proper degree of luminescent signal (at least a 5 fold signal to noise ratio, and within the linear range of detection for the plate reader you are using). This can be tested by measuring signal from different amounts of lysate (between 5 and 80 μl) and plotting the corresponding luminescent values. Housekeeping and target genes may require different volumes of lysate in the same experiment.
    9. Top off each well to 100 μl with DLM (no Proteinase K).
    10. Seal the plate very tightly with an aluminum plate seal (provided with the kit) and centrifuge for 1 min at 240 x g at room temperature.
    11. Incubate over night at 55 °C in a forced air oven.
    12. The next day perform signal amplification as recommended by Affymetrix.
    13. Read the luminescence using a microplate reader setup for a 200-500 msec reading time per well.
    14. Analyze the data.

Data analysis

Note: Data are processed using Microsoft Office Excel and analyzed using GraphPad Prism 7 software.

  1. For each plate and each individual probeset, average the values obtained in the DLM (blank) wells and subtract the values from all sample wells.
  2. To normalize the target gene expression, divide the target gene luminescent value by the housekeeping gene luminescent value.
  3. Average the normalized untreated cell values.
  4. To calculate the percent of target gene expression relative to untreated cells, divide each individual normalized well by the average of the untreated wells and multiply by 100.

  5. If compounds are tested in replicate, average the replicates and calculate the standard deviation.
  6. For dose response analysis, graph concentration-dependent IC50 curves using a non-linear regression curve fit, log(inhibitor) vs. response–variable slope (four parameters). If necessary, set the lower limit of the curve at zero, and the upper limit of the curve at 100.
  7. Compare efficacy of target sequences to the non-targeting control (NTC) to determine statistical significance. Differences in all comparisons are considered significant at P-values less than 0.05 compared with the NTC group.


  1. After dissociation, cells look round if the trituration was gentle.
  2. Cells should show signs of making neurites by two hours after plating. If they are still round without neurites on the second day, they will probably not grow.
  3. Cells should be plated as a single cell suspension. If cells are doublets or triplets, they tend to grow in clumps, which will affect the data of the experiment, as well as the cell growth.
  4. Pay attention to the neuronal medium lot. Lot to lot variability can affect the clumpiness, growth, and viability of the cells.
  5. Particular attention should be given to the hsiRNA modification pattern. Functionally efficient hsiRNA requires extensive chemical stabilization. Chemical modification of the ribose with 2’-O-methyl and 2’-fluoro results in a significant increase of the hsiRNA-resistance to nuclease degradation, providing higher stability in vitro and in vivo (Byrne et al., 2013; Alterman et al., 2015). Unmodified or partially modified hsiRNAs will undergo nuclease degradation, affecting functional efficacy in culture.
  6. We have observed that at high concentrations of hsiRNA (2 µM and up) induces the formation of cell clumps.
  7. If the cells are clumping or pulling away from the edge of the well (aggregating at the center of the well) later in the hsiRNA incubation period, this is a sign of unhealthy treatment or plating conditions.


Note: For all recipes and at any step of the procedure use Milli-Q-purified water.

  1. Papain/DNase solution
    1. Dissolve Papain in 2 ml Hibernate E and 1 ml PBS and mix gently by inverting the vial 2-3 times (Do not vortex)
    2. Separately, suspend DNase in 0.5 ml Hibernate E and gently invert the vial 2-3 times
    3. The final Papain solution contains 0.25 ml of suspended DNase mixed in 3 ml of Papain solution
  2. Plating medium
    NbActiv4 supplemented with 2.5% FBS
    For 50 ml of plating medium, add 1.25 ml of 100% FBS in 48.75 ml of NbActiv4
    Alternatively, use NeuralQTM medium supplemented with GS21TM supplement (50x), 0.5 mM dipeptide Ala-Gln and 2.5% FBS
  3. Feeding medium
    NbActiv4 supplemented with anti-mitotics, 0.484 μl/ml of 5’UTP and 0.2402 μl/ml of 5’FdU
    For 50 ml of NbActiv4, add 24.2 μl of 5’UTP and 12 μl of 5’FdU
    Alternatively, use NeuralQTM medium supplemented with GS21TM supplement (50x), 0.5 mM dipeptide Ala-Gln, and anti-mitotics 0.484 μl/ml of 5’UTP and 0.2402 μl/ml of 5’FdU


We thank all the members of the Khvorova and Aronin Laboratories, NIH and CHDI Foundation Inc. for helpful discussions. Specifically, we would like to thank Kathryn Chase in the Aronin Laboratory, from whom the primary neuron isolation protocol was originally adapted. This work is supported in part by grants from the NIH UH2-TR000888 and UH3-4UH3TR000888-03, NIH NS38194 and CHDI Foundation (Research Agreement A-6119). The authors declare no conflict of interest.


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原代神经元是鉴定用于治疗神经变性疾病的治疗性寡核苷酸的理想细胞系统。然而,由于原代细胞的敏感性,使用经典方法转染小干扰RNA(siRNA)是费力的,并且通常显示低效率。寡核苷酸化学的最新进展使得稳定和疏水修饰的小干扰RNA(hsiRNA)的发展成为可能。这种新型的寡核苷酸治疗剂显示出非常有效的自我传递性质,并且在体外和体内支持有效和持久的效果。我们开发了高通量的体外测定法来鉴定和测试原代神经元培养物中的hsiRNA。为了简单,快速,准确地量化数百个hsiRNA的mRNA沉默,我们使用QuantiGene 2.0定量基因表达测定法。这种高通量,96孔板测定法可以直接从样品裂解液中定量mRNA水平。在这里,我们描述了一种制备96孔板格式的小鼠原代皮质神经元的短期培养物用于寡核苷酸治疗剂的高通量测试的方法。该方法支持在短短两周内测试hsiRNA文库和鉴定潜在的治疗方法。我们详细介绍了从初级神经元准备到数据分析的高通量测定工作流程的方法。该方法可以帮助鉴定用于治疗各种神经疾病的寡核苷酸治疗剂。
【背景】寡核苷酸治疗剂代表了通过沉默突变蛋白的表达,可以靶向任何遗传定义的病症的新一类药物。具体地,siRNA是负载于RNA诱导的沉默复合体(RISC)中的双链寡核苷酸,并且可以在mRNA翻译之前使mRNA沉默。然而,未修饰的siRNA是不稳定的,并且不能在没有阳离子脂质制剂的帮助下进入细胞,其可能对原代细胞如神经元有毒性。在本协议中,我们使用自我递送,疏水修饰的siRNA(hsiRNA)进行mRNA沉默。最近在寡核苷酸化学方面的进展使得这些稳定的hsiRNA的设计促进了细胞内化,有效进入RISC以及有力击倒靶基因(Byrne等,2013; Alterman et al。,2015; Ly et al 2017年)。这些化合物对所有糖和硫代磷酸酯主链修饰都含有2'-O-甲基和2'-氟修饰;寡核苷酸通常与疏水部分如胆固醇缀合,以支持膜结合和细胞内化而无毒性。这种新型化合物为研究人员提供了一种直观的方法,可以在生物相关性和难以转染的原代皮层神经元的背景下沉默各种基因(Alterman等,2015)。
   今天,在药物发现的早期阶段,大多数高通量测试在基于细胞的铅测定中进行,用于铅寡核苷酸鉴定和验证。基于细胞的测定改进和加速药物筛选,提供比生物化学测定更为相关的体内生物信息,并减少动物测试的需要。在初级神经元中进行的高通量测定已经成为发现用于治疗神经退行性疾病(例如亨廷顿氏病(HD),肌萎缩性侧索硬化症(ALS)或阿尔茨海默病)(AD))的新疗法的有力工具(Sharma et al。 ,2012)。使用原代神经元的基于细胞的测定为克隆神经元细胞系提供了更自然(相关)的研究神经变性疾病的环境。将基于寡核苷酸的治疗剂转染到原代神经元中通常依赖于诸如电穿孔的方法(Mertz等人,2002; Gresch等人,2004; Zhang等人,2016),病毒转导(Naldini等人,1996; Hughes et al。等等,2002; Janas等人,2006)或脂质介导的转染(Ohki等人,2001; Dalby等人,2004; Zhang等人,2016)。然而,这些方法可能费力,显示效率低,并诱导细胞毒性。因此,hsiRNA的自我传递性质代表了在复杂细胞模型(如原代神经元)中鉴定体外新引线的有效方法。我们最近证实,hsiRNA在治疗后数秒钟内有效地结合神经元细胞膜,进入细胞,并且在体外,原代神经元和体内,在小鼠脑中诱导有力的基因沉默,所有这些均不使用转染试剂( Alterman等,2015; Ly et al。,2017)。
   我们的实验室建立了一个快速的高通量平台,以96孔格式体外鉴定和验证原代神经元中的hsiRNA引线。为了量化siRNA在siRNA上的量,我们使用QuantiGene 2.0支链DNA(bDNA)测定法,以高通量,96孔板为基础的mRNA定量测定。该技术设计用于直接定量来自样品裂解物的靶mRNA,而不需要纯化RNA,从而最小化样品操作(Kern等,1996; Coles等,2015)。该测定使得能够准确和精确地检测甚至低丰度mRNA,最小化实验变异性和错误(Collins等人,1997; Canales等人,2006)。自我传递hsiRNA和mRNA的高通量定量的组合加速了原代神经元中有效寡核苷酸的鉴定。

关键字:原代皮质神经元, siRNA, 筛查, 分支DNA, QuantiGene 2.0


  1. 聚-L-赖氨酸预涂组织培养处理的96孔板(Corning,目录号:356516)
  2. 深96孔无菌聚丙烯板(Corning,Axygen ,目录号:391-04-062)
  3. 提示(0.2μl至1 ml)(VWR)
  4. 血清学移液器,单独包裹(从5ml到50ml)(Costar)
  5. 消火抛光巴斯德吸管用棉塞(内部制造)
  6. 组织培养处理10厘米盘(康宁,目录号:430167)
  7. 50ml锥形离心管(Corning,Falcon ®,目录号:352097)
  8. 15ml锥形离心管(Corning,Falcon ®,目录号:352098)
  9. 1.7ml微量离心管(Genesee Scientific,目录号:22-282)
  10. 1 ml无菌注射器(BD,目录号:309659)
  11. 粘合板密封(VWR,目录号:60941-126)
  13. 胆固醇结合的hsiRNA(内部设计和生产)
  14. 冰冷块(Koolit ®制冷剂)(Cold Chain Technologies,目录号:305F)
  15. 聚-L-赖氨酸(Sigma-Aldrich,目录号:P4707)
  16. 磷酸盐缓冲盐水(PBS)(Mediatech,目录号:21-031-CV)
  17. 200型乙醇(Decon Labs,目录号:2701)
  18. DMEM细胞培养基(Mediatech,目录号:10-013-CV)
  19. Hibernate E(BrainBits,目录号:HE)
  20. DNase I(Worthington,目录号:54M15168)
  21. 木瓜蛋白酶(Worthington,目录号:54N15251)
  22. 台盼蓝染色溶液(Thermo Fisher Scientific,Gibco TM,目录号:15250061)
  23. QuantiGene 2.0测定试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:QS0011)
  24. QuantiGene 2.0探针组(按基因变化)
  25. 裂解混合物(Thermo Fisher Scientific,Affymetrix TM,目录号:13228)
  26. 蛋白酶K(Thermo Fisher Scientific,Affymetrix TM,目录号:QS0103)
  27. Neurobasal medium NbActiv4(BrainBits,catalog number:Nb4-500)
  28. 胎牛血清(FBS)(Mediatech,目录号:35-010-CV)
  29. 补充有GS21 补充剂(50x)(Sigma-Aldrich,目录号:G0800)的NeuralQ TM(Sigma-Aldrich,目录号:N3100),0.5mM二肽Ala -Gln(Sigma-Aldrich,目录号:A8185)
  30. 5'UTP(Sigma-Aldrich,目录号:U6625)
  31. 5'FdU(Sigma-Aldrich,目录号:F3503)
  32. 木瓜蛋白酶/ DNase溶液(参见食谱)
  33. 电镀介质(见配方)
  34. 饲养介质(见食谱)


  1. 0.5微升至1毫升的微量移液器(Labnet International,型号:BioPette Plus)
  2. 10通道至300μl的多通道(8或12通道)微量移液管(Eppendorf,型号:Research ®)
  3. 组织培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heracell TM 150i)
  4. 连接真空的生物安全柜(Thermo Fisher Scientific,Thermo Scientific TM,型号:1300系列II类,A2型)
  5. 本生灯
  6. 发芽子500(Braintree Scientific,型号:Germinator 500,目录号:GER 5287-120V)
  7. 37°C的水浴(Fisher Scientific,型号:2332型)
  8. 4°C冰箱
  9. Microscissors(Fine Science Tools,目录号:14060-10和14002-12)
  10. 一套两个镊子(精细科学工具,目录号:11251-30)
  11. 解剖显微镜(Motic,型号:SMZ168系列)
  12. 吸管(Drummond Scientific,型号:Portable Pipet-Aid ® XP)
  13. 组织培养相差倒置显微镜(Motic,型号:AE2000)
  14. 血细胞计数器(0.1000-0.0025mm 2 )(Neubauer)
  15. μPlate载体(Beckman Coulter,型号:SX4750)
  16. 自动洗板机(BioTek Instruments,型号:ELx405)
  17. 冷藏式摆动转子台式离心机(Beckman Coulter,型号:Allegra X-15R)
  18. Allegra X-15R转子(Beckman Coulter,型号:SX4750)
  19. 读卡器分光光度计(Tecan Trading,型号:Infinite M1000 Pro)


  1. Microsoft Office Excel(Microsoft Office)
  2. GraphPad Prism 7软件(GraphPad Software)


  1. 组织培养的聚-L-赖氨酸包被96孔板
    1. 为了涂覆平板,使用多通道微量移液管将100μl的0.01%聚-L-赖氨酸转移到每个孔中。
    2. 在组织培养箱37℃孵育过夜。
    3. 第二天,在解剖之前,使用真空连接的巴斯德吸管(或200μl多通道微量吸管)吸出涂层溶液,并用100μlPBS冲洗三次。
    4. 取出最后的洗涤液,让板在生物安全柜中干燥。

  2. 准备棉塞,火抛光巴斯德吸管
    1. 使用本生灯,通过将毛细管定向在火焰中2-3毫米的移液管快速旋转,对棉塞堵塞的巴斯德吸管末端进行抛光。
    2. 检查毛细管的开口,确保边缘圆形,孔不会太小。如果孔太小,丢弃移液管,因为它会在研磨过程中抛出神经突(图1)。
    3. 使用前,将巴斯德吸液管高压灭菌。

      图1.消防抛光移液器。 A.棉花塞,火抛光移液器; B.从左到右:未抛光的尖端(边缘太尖锐,白色箭头),正确抛光的尖端(边缘光滑,绿色箭头),过度抛光的尖端(开口太小,红色箭头) ; C.与B相同
  3. 分离小鼠原代皮质神经元(图2)


    1. 对具有CO 2的怀孕小鼠进行安乐死,然后颈部脱位。
    2. 将鼠标放在她的背上,用70%乙醇清洁腹部表面以对该区域进行消毒。
    3. 使用微型剪刀,在腹部肌肉中进行垂直切口,打开小鼠腹腔。使用镊子,仔细取出胚囊并在细胞培养基(DMEM,冰冷,无添加剂)中冲洗。
    4. 将胚胎囊置于含有10毫升冰冷DMEM的10厘米盘中
    5. 使用微型剪刀和镊子仔细打开胚胎囊,取出胚胎并转移到含有10毫升冰冷DMEM的10厘米盘中。
    1. 将镊子放置在胚胎头骨后面,向鼻前方向前推。
    2. 将镊子放在头部的两侧,大脑下方,并小心地将脑部从头骨中抬出(视频1)。

      Video 1. Removal of the brain from the skull

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    3. 将大脑转移到含有10毫升冰冷的冬眠E的10厘米盘中
    4. 在解剖显微镜下,将大脑转移到10厘米盘中的Hybernate E的单液滴,以便于进入。
    5. 在解剖显微镜下,使用镊子仔细地从每个脑半球的上表面剥离脑膜。去除脑膜后,皮质会脱离大脑(图3B)
    6. 使用镊子,仔细分离皮质与大脑。

      图3.去除脑膜以释放皮质。 A.从头骨中移除个体大脑;去除脑膜后,从一个半球的皮层; C.一个半球的皮质(虚线)。皮质区转移到木瓜蛋白酶/ DNA酶(实线)
    7. 将皮质的上部,外部部分(图3C)转移到含有1ml预温热木瓜蛋白酶/ DNA酶溶液的1.5ml微量离心管中,并收集其余皮质,同时尽可能多地吸收污染物。
    8. 孵育管开放,在无菌10厘米的培养皿中37分钟,以溶解组织。这使得组织在保持无菌环境的同时呼吸。
    1. 将管子立起放置几分钟,以使皮质在管底部沉淀。
    2. 在不干扰皮质的情况下,使用1 ml微量移液缓慢小心地去除木瓜蛋白酶/ DNA酶溶液(确保尽可能多地除去木瓜蛋白酶/ DNA酶溶液),并加入1ml电镀培养基。
    3. 使用调节至最低速度的移液器,通过棉花塞,火焰抛光的巴斯德移液管,以平缓和一致的移动方式上下移动细胞来分离细胞。避免气泡的形成是很重要的。研磨组织直至培养基稍微浑浊,并且没有剩余的组织块可见(视频2)。

      Video 2. Trituration of cortices

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    4. 将细胞转移到15ml锥形管中并加入4ml电镀培养基。为了确保细小的细胞块被破碎,通过使用调节至最低速度的移液器,通过棉花塞,火焰抛光的巴斯德移液管,轻轻地和一贯地上下移动多次,进行额外的研磨。
    5. 让溶液静置2分钟,使任何碎屑沉淀到15 ml管的底部
    6. 将含有单细胞神经元悬浮液的上清液转移到新的15 ml管中。
    7. 将小滴细胞转移到组织培养处理的培养皿中,并使用组织培养显微镜(10倍放大)观察是否存在单细胞悬浮液(图4)。如果没有,如前所述,使用棉花塞,火抛光的巴斯德吸管进行另一次研磨。


    8. 使用台盼蓝染色液和血细胞计数器,计数存在于细胞悬浮液中的活细胞。加入10μl细胞至90μl台盼蓝染色液中,并通过移液轻轻混匀。将10μl等分试样转移到血细胞计数器,并计数血细胞计数器所有四个象限的活细胞。平均四个数字乘以1 x 10 5 。该数目对应于每ml培养基的细胞数。平均而言,每只小狗将产生〜5-10×10 6个细胞。
    9. 将细胞转移到50ml锥形管中,并用预热的培养基将活细胞的密度调节至1×10 6细胞/ ml。例如,如果细胞浓度在5ml中为5×10 6细胞/ ml,则加入20ml的电镀培养基以获得最终细胞浓度为1×10 6细胞/细胞/ ml,最终体积为25 ml
    10. 关闭并倒置管子以确保细胞悬液的充分混合。将细胞转移到50ml试剂储存器。
    11. 使用200μl多通道微量移液管,将100μl细胞(1×10 5个细胞)转移到聚-L-赖氨酸预包被的96孔板的每个孔中。为了在每个孔中种下相同数量的细胞,在每次转移到96孔板中之前通过上下移液来混合储存器中的细胞。
    12. 在组织培养箱中37℃,5%CO 2孵育板过夜。
    13. 第二天,使用多通道微量移液管,每孔缓慢加入100μl饲养培养基(含有抗精神病药),以防止非神经元细胞的生长。小心不要打扰细胞。
    14. 每3-4天,用新鲜准备的饲养介质取代一半体积的培养基。使用多通道微量移液管,从每个孔中缓慢移除100μl培养基,而不要接触孔的底部,以免扰乱细胞。非常轻轻地添加100μl新鲜制备和预热的饲养培养基,而不会干扰细胞。
    15. 储存在组织培养箱37℃。必要时重复进料步骤。

  4. 用寡核苷酸治疗治疗原发性神经元
    在饲养培养基中制备寡核苷酸稀释液。仔细设计您的平板图以包括感兴趣的寡核苷酸(hsiRNA Target ),非靶向控件(hsiRNA NTC sup>)和未处理的细胞。化合物以最终浓度的两倍制备,以便可将50μl化合物溶液加入到50μl条件培养基中。确保准备足够的化合物以处理所需重复次数的细胞。为了本协议的目的,我们描述了每种化合物的三个重复孔的制备和1.5μMhsiRNA的最终浓度。
    1. 在深96孔板中,通过将含有抗神经系统的新鲜饲养培养基中的库存hsiRNA稀释至最终浓度为3μM(最终浓度为1.5μM)的2×hsiRNA主混合物,最终体积为200μl微升。
    2. 用微量移液器轻轻上下混合。
    3. 使用微量移液管,轻轻地从神经元中移除整个条件培养基,并立即将50μl条件培养基加入每个孔中(您可以将条件培养基合并到无菌试剂容器中,以便将其吸入板中)。一次只能从一排或两行中取出培养基,以防止细胞干燥。为了避免干扰细胞,用尖端接触孔的边缘缓慢移液介质。该步骤旨在控制在37℃孵育期间可能发生的任何蒸发,并确保所有细胞用相同浓度的hsiRNA处理。
    4. 使用多通道微量移液管和每一行的清洁尖端,将50μl的2x hsiRNA主混合物从深96孔板转移到细胞培养板的每个重复孔中,最后的体积为每孔100μl。
    5. 将培养板在组织培养箱中37℃孵育。在一周的温育中,用100μl新鲜制备的饲养培养基(处理3-4天)喂养细胞一次。然后在治疗一周后溶细胞。该步骤确保在实验期间不会从孔中除去hsiRNA。对于长期实验,细胞需要不止一次进食,从细胞培养板200μl中取出100μl条件培养基,加入100μl新鲜培养基。继续这种3-4天的喂养模式。

  5. mRNA定量
    使用QuantiGene 2.0bDNA测定(Affymetrix)定量mRNA。以下步骤中使用的所有体积均为一块。使用干净的血清移液管,在干净的50ml锥形管中准备用于此程序的所有溶液。
    1. 在50ml锥形管中,制备补充有100μl的溶液的稀释的裂解混合物(DLM; 1:2v / v,裂解混合物/水,例如,10ml裂解混合物:20ml水) 50μg/μl蛋白酶K(随试剂盒提供),倒入50ml容器中。稀释的裂解混合物应为每个实验新鲜准备。将稀释的溶解混合物储存在冰箱中将导致沉淀物形成
    2. 从板中取出所有生长培养基。
    3. 使用300μl多通道移液管,在96孔培养板的每个孔中加入250μl稀释的裂解混合物。有足够的裂解液来进行目标和对照mRNA的定量。
    4. 要细胞裂解,轻轻移动上下,避免形成气泡。每行之间使用干净的提示。
    5. 在强制通风烘箱中,在55℃下孵育30分钟。
    6. 使用300μl多通道移液器设置在150μl,通过轻轻地上下移动来彻底混合细胞裂解物,避免形成气泡。
    7. 加入20μl探针组,根据试剂盒方案稀释到捕获板的每个孔(随试剂盒提供)。
    8. 使用100μl多通道移液器,将适量的每个裂解液转移到捕获板。避免接触涂层板的底部。对于背景井,只需添加DLM(无蛋白酶K),而不是细胞裂解液。在使用QuantiGene 2.0试剂盒测试新基因之前,您将需要验证裂解物的体积,以获得适当程度的发光信号(至少5倍的信噪比,并且在线性检测范围内你正在使用的读卡器)。可以通过测量来自不同量的裂解物(5至80μl)的信号并绘制相应的发光值来测试。在同一个实验中,家务和目标基因可能需要不同体积的裂解物
    9. 用DLM(无蛋白酶K)将每个孔上清至100μl。
    10. 用铝板密封(随套件提供)非常紧密地密封板,并在室温下以240×g离心1分钟。
    11. 在强制空气烘箱中在55°C下孵育过夜。
    12. 第二天执行Affymetrix推荐的信号放大。
    13. 使用酶标仪阅读器读取每个孔的读数时间为200-500毫秒。
    14. 分析数据。


注意:使用Microsoft Office Excel处理数据,并使用GraphPad Prism 7软件进行分析。

  1. 对于每个板和每个单独的探针组,平均在DLM(空白)孔中获得的值,并从所有样品孔中减去值。
  2. 为了使目标基因表达正常化,将目标基因发光值除以管家基因发光值
  3. 平均归一化未处理的细胞值。
  4. 为了计算相对于未处理细胞的靶基因表达的百分比,将每个单独的标准化均匀化除以未处理孔的平均值,并乘以100.

  5. 如果化合物在复制中进行测试,则平均复制并计算标准偏差
  6. 对于剂量反应分析,使用非线性回归曲线拟合,log(抑制剂)对响应变量斜率(四个参数)的图表浓度依赖性IC 50曲线。如果需要,将曲线的下限设置为零,曲线上限为100.
  7. 比较目标序列与非靶向对照(NTC)的功效以确定统计学显着性。与NTC组相比,所有比较的差异被认为在P - 值小于0.05的显着性。


  1. 解离后,如果研磨温和,细胞看起来会圆润。
  2. 细胞应在电镀后2小时内显示发生神经突的迹象。如果第二天仍然没有神经突,他们将不会长大。
  3. 细胞应作为单细胞悬浮液进行电镀。如果细胞是双峰或三胞胎,则它们倾向于以块状生长,这将影响实验的数据以及细胞生长。
  4. 注意神经元介质很多。批次变化可能会影响细胞的结块,生长和活力。
  5. 应特别注意hsiRNA修饰模式。功能高效的hsiRNA需要广泛的化学稳定性。用2'-O-甲基和2'-氟进行核糖的化学修饰导致hsiRNA对核酸酶降解的抗性的显着增加,在体内提供更高的稳定性和体内< (Byrne等人,2013; Alterman等人,2015)。未修饰或部分修饰的hsiRNA将经历核酸酶降解,影响培养物的功效
  6. 我们已经观察到,在高浓度的hsiRNA(2μM及以上)诱导细胞团的形成。
  7. 如果在hsiRNA潜伏期后期,细胞会从井的边缘聚集或拉出(在孔的中心聚集),这是不健康的治疗或电镀条件的迹象。



  1. 木瓜蛋白酶/ DNase溶液
    1. 将木瓜蛋白酶溶解在2ml Hibernate E和1ml PBS中,通过倒置小瓶2-3次轻轻混合(不要旋涡)
    2. 另外,将DNA酶悬浮在0.5ml Hibernate E中,轻轻倒置小瓶2-3次
    3. 最后的木瓜蛋白酶溶液含有0.25ml悬浮的DNase,混合在3ml木瓜蛋白酶溶液中
  2. 电镀介质
    中加入1.25ml 100%FBS 或者,使用补充有GS21 TM补充剂(50x),0.5mM二肽Ala-Gln和2.5%FBS的NeuralQ TM 培养基
  3. 饲养培养基
    补充有抗神经元的NbActiv4,0.484μl/ ml的5'UTP和0.2402μl/ ml的5'FdU
    或者,使用补充有GS21 TM补充剂(50x),0.5mM二肽Ala-Gln和抗 - mitotics的NeuralQ 培养基0.484μl/ ml 5'UTP和0.2402 μl/ ml的5'FdU


我们感谢Khvorova和Aronin实验室,NIH和CHDI基金会公司的所有成员进行有益的讨论。具体来说,我们要感谢Aronin实验室的Kathryn Chase,其中主要的神经元分离方案最初来自该实验室。这项工作部分由NIH UH2-TR000888和UH3-4UH3TR000888-03,NIH NS38194和CHDI基金会(研究协议A-6119)的资助部分支持。作者宣称没有利益冲突。


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引用:Alterman, J. F., Coles, A., Hall, L. M., Aronin, N., Khvorova, A. and Didiot, M. (2017). A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics. Bio-protocol 7(16): e2501. DOI: 10.21769/BioProtoc.2501.