User Guide
Quick-Neuron™ Sensory - mRNA Kit (Large)
Catalog Number: SS-mRNA-L

Introduction
The Quick-Neuron™ Sensory - mRNA Kit (Large) facilitates rapid and efficient differentiation of human iPS or ES cells into a
population of sensory neurons in just 10 days. Our proprietary transcription factor-based stem cell differentiation method
uses synthetic mRNAs to produce highly pure populations of neurons without a genetic footprint. Quick-Neuron™ Sensory
differentiated cell cultures display typical neurite outgrowth and express a variety of neuronal markers, such as tubulin beta
3 class III (TUBB3) and a variety of sensory neuron markers such as sodium voltage-gated channel alpha subunit 9(SCN9A/Nav1.7), peripherin (PRPH), islet-1 (ISL1), and brain-specific homeobox/POU domain protein 3A(BRN3A/POU4F1). When handled and maintained according to the instructions in this user guide, sensory neurons are
viable long-term and are suitable for a variety of characterization and neurotoxicity assays.
Scale:The Quick-Neuron™ Sensory - mRNA Kit (Large) contains a set of reagents for use with a total of 6
wells of a 6-well plate.

Related Products:

Quick-Neuron™ Sensory - mRNA Kit (Small), Catalog Number: SS-mRNA-S
Quick-Neuron™ Sensory - Human iPSC-derived Neurons, Catalog Number: SS-mRNA-CW
Quick-Neuron™ Sensory - Maintenance Medium, Catalog Number: SS-MM

Contents
Upon receipt, store the reagents at the temperatures indicated in the table below. All reagents are shipped on dry ice.
Contents

VolumeStorageThaw

QNS-mRNA-P

4 x 33 μl-80°C

On iceComponent N

4 x 840 μl-20°C or -80°C

On ice or 4°C

Component S1

5 x 22 μl-20°C or -80°C

On ice or 4°C

Component P

2 x 50 μl-20°C or -80°C

Room temperature

Condition of Use
This product is for research use only. It is not approved for use in humans or for therapeutic or diagnostic use.

Technical Support
For technical support please refer to the FAQ on our website.
You may also contact us at cs@elixirgensci.com or call +1 (443) 869-5420 (M-F 9am-5pm EST).

Last revised: August 04, 2023

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1Required Consumables
Item

VendorCatalog Number

6-well tissue-culture-treated polystyrene plate (e.g., Corning
Costar Flat Bottom Cell Culture Plates)

Fisher Scientific07-200-80(Optional) 24-well tissue-culture-treated polystyrene plate(e.g., Corning Costar Flat Bottom Cell Culture Plates)

Fisher Scientific07-200-740(Optional) 96-well tissue-culture-treated polystyrene plate(e.g., Thermo Scientific™ 96 Well Black/Clear Bottom Plate)Fisher Scientific12-566-70

Lipofectamine MessengerMAX

ThermoFisher

LMRNA001

Opti-MEM I Reduced Serum Medium

ThermoFisher

31985062

DMEM/F12

ThermoFisher

21331020

Neurobasal Medium

ThermoFisher

21103049

GlutaMAX

ThermoFisher

35050061

Penicillin-Streptomycin

ThermoFisher

15140122

StemFit Basic04 Complete Type, or
StemFit AK02N, or
StemFlex Medium

Elixirgen Scientific
TaKaRaThermoFisher

ASB04-C, or
AK02N, orA3349401

iMatrix-511 silk

Elixirgen Scientific

NI511S

TrypLE Select Enzyme (1X)

ThermoFisher

12563011

0.02% EDTA in DPBS

Sigma-Aldrich

E8008-100ML

0.01% Poly-L-Ornithine

Sigma-Aldrich

P4957-50ML

Extracellular Matrix such as
Laminin Mouse Protein, Natural, or
Geltrex Basement Membrane Matrix

ThermoFisher

23017015 orA15696-01

Phosphate-buffered saline (without Ca++ Mg++)*

ThermoFisher

20012050

ROCK inhibitor Y27632

Selleckchem

S1049

Dimethyl sulfoxide (DMSO)

Sigma-Aldrich

D2650

InvivoGen

ant-pr-1

Puromycin (10 mg/ml)* PBS should be used at room temperature unless otherwise specified.

Source hPSC Culture Conditions
The Quick-Neuron™ Sensory - mRNA Kit (Large) gives the best differentiation results when source human pluripotent stem
cells (hPSCs) have been maintained in StemFit® Basic04, StemFit® AK02N, StemFlex™ Medium, or other similar culture
media which enable the maintenance of cultures by single-cell passaging. If iMatrix-511 silk is routinely used as a coating
substrate, prepare culture dishes or wells precoated with 0.25 μg/cm2 iMatrix-511 silk diluted in 2 ml chilled PBS per well ordish for this kit.
● The protocols and reagents for StemFit® Basic04 and iMatrix-511 silk culture conditions are available at Elixirgen
Scientific (Catalog Numbers: ASB04-C, NI511S).
● Differentiation should not be performed until the cells are at least 14 days post-thaw.

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●
●

We recommend preparing a minimum of 4.6 x 106 viable hPSC for use with this kit. This is usually obtained by using
2-3 wells of a 6-well plate at 50-70% confluency.
For optimal differentiation, hPSC confluency should be around 50% to 70%. Do not use wells more than 90%
confluent.

Drug Selection
Users should perform a puromycin kill curve for their cells to determine the minimum concentration required to kill all
non-treated cells within ~60 hours. Based on Elixirgen Scientific’s internal tests, the appropriate concentration ranges
between 0.5 and 2 µg/ml. We recommend maintaining 2 wells of untransfected iPSC (with standard Stemfit conditions),
alongside the transfected wells, until after puromycin selection is performed. Treat 1 of those wells with puromycin at yourselected concentration so as to confirm that the puromycin is effective at killing the untransfected cells in your experiment.

Workflow
Note: This protocol assumes that Day 0 is a Monday.

* From Day 10, users may maintain differentiated neurons in the medium best suited for their needs, though we recommend
Quick-Neuron™ Sensory - Maintenance Medium, Catalog Number: SS-MM.

Preparation
Important Note! For the best possible delivery of QNS-mRNA-P into cells, we recommend Lipofectamine MessengerMax.
If users prefer another transfection reagent, please make sure that the reagent provides a transfection efficiency of ≥80%
prior to using this kit. QNS-mRNA-P mixed with Lipofectamine MessengerMax must be immediately applied to cultures andcannot be stored.
10 mM ROCK inhibitor Y27632 (iROCK)
1. Dissolve 10 mg ROCK inhibitor Y27632 in 3.12 ml DMSO.
2. Make aliquots of a convenient volume (e.g., 100 μl).
3. This solution, hereafter referred to as iROCK, can be stored at -20°C.
StemFit Basic04 Complete Type (Medium S)*
1. Thaw StemFit Basic04 Complete Type bottle overnight or multiple nights at 4°C.
2. Make aliquots of a convenient volume (e.g., 40 ml).
3. This solution, hereafter referred to as Medium S, can be stored at -80°C.
*Medium S can be substituted with StemFit AK02N or StemFlex.

0.5X TrypLE Select with EDTA (Solution D1)
1. Mix 1 ml TrypLE Select Enzyme (1X) with 1 ml 0.02% EDTA in DPBS.
2. This mixture, hereafter referred to as Solution D1, can be stored at 4°C for 2 weeks.
0.002% Poly-L-Ornithine solution (ornithine)
1. Take 700 μl 0.01% Poly-L-Ornithine solution and mix it with 2.8 ml PBS.
2. The 0.002% Poly-L-Ornithine solution, hereafter referred to as ornithine, can be stored at 4°C for up to 2 weeks.
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1 mg/ml laminin stock solution (laminin)
1. Thaw Laminin Mouse Protein, Natural and chill PBS at 4°C or on ice.
2. Mix the Laminin Mouse Protein, Natural and PBS to make the 1 mg/ml stock solution, hereafter referred to aslaminin.
○ Laminin concentration varies by lot, so use the number specified on the vial or CoA when making yourcalculations.
3. Make aliquots of a convenient volume (e.g., 35 μl) and store at -20°C.
Medium N(S1)1.

Prepare Medium N(S1) using the reagents listed in the table below.
○ Thaw Components N and S1 for 20-30 minutes at the temperatures indicated in the “Contents” table on
page 1.
○ Warm all other reagents at room temperature for 20-30 minutes.
○ Tap each Component tube 3 times and then briefly spin all tubes down before use.
○ Keep Medium N(S1), and any subsequent media made with it, protected from light.
○ Store Medium N(S1) for up to 2 weeks at 4°C.
○ Leftover Components N and S1 can be discarded or saved at 4°C for up to two weeks.
Reagents

VolumeDMEM/F12

49.6 ml

Neurobasal

49.6 mlGlutaMAX

520 μlPenicillin-Streptomycin (10000 units/ml; 100x)

1 ml

Component N

3.2 mlComponent S1

104 μlDay -3~1 hour

Note: This protocol assumes that Day 0 is a Monday so Day -3 is Friday.
Plate Preparation1.

Prepare diluted iMatrix-511 silk by mixing together the following components in a 15 ml conical tube.
○ Keep iMatrix-511 silk on ice.
○ Make sure chilled PBS is used for this mixture.
Reagents

VolumeiMatrix-511 silk

43.2 µl

Chilled PBS

13 ml

2. Add 2 ml diluted iMatrix-511 silk to each new well of a new 6-well plate.
3. Incubate the plate at 4°C for 3 days.
Note: For best results we recommend precoating the plate 1 day or up to a week before use and keeping at 4°C.
Alternatively plates can be precoated on Day 0 and placed at 37°C for at least 2 hours before use.

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4Day 0~4 hours

Note: This protocol assumes that Day 0 is a Monday and that user’s hPSCs were already used with a small size kit(Catalog number: SS-mRNA-S) so that users are familiar with the experimental process and have optimized conditions
for their particular cells.
Plating
IMPORTANT! Source hPSC wells should be no more than 50-70% confluent thus requiring a minimum of 2 wells to
begin differentiation.
Determine the number of wells required to get 4.6 x 106 cells from the source hPSC 6-well plate.
NOTE: Cells will be plated in a new 6-well plate at 3 densities (0.6 x 106 cells, 0.7 x 106 cells, and 0.8 x 106 cells),
with 2 wells per density.
2. Prepare Medium iS by mixing together the following components in a 15 ml conical tube.
○ Warm Medium S, iROCK, and Solution D1 at room temperature for at least 1 hour protected from light.
○ The rest of Medium S should be stored at 4°C for later use.
1.

Required medium volume based on # of wells of
a 6-well plate
Reagents

2 wells

3 wells

Medium S

12.7 ml

15.5 ml

iROCK

12.7 µl15.5 µl

3. Aspirate old medium from hPSC culture and add 1.5 ml of Medium iS to each well.
4. Incubate the culture at 37°C, 5% CO2 for 1 hour before harvesting cells.
○ This is to decrease cell death on Day 1 and minimize the loss of cells.
5. Aspirate old medium from hPSC culture and add 2 ml PBS.
6. Rock the plate 3 times, aspirate PBS from the culture, and add 300 μl of the cell dissociation reagent Solution D1.
Keep the rest of Solution D1 at 4°C for use on Day 4.
7. Incubate the culture plate at 37°C, 5% CO2 for 5 minutes. If all the cells are not rounded under a microscope,
continue to incubate at 37°C, 5% CO2 in 1-2 minute increments (see images below).

8. Carefully pipet out Solution D1 from the culture and add 1 ml Medium iS to the well.
○ Follow Steps 8-10 one well at a time if multiple wells are used.
9. Disperse the medium over the bottom surface of the well by pipetting 8-15 times to detach cells.
10. Using the same pipet tip, collect the cell suspension in a 15 ml tube.
11. Count cells and determine viability.
12. Take out the volume of the cell suspension needed for 2 wells of each cell density, according to the note in step 1,
and include 10% extra. Place each in a new tube labeled with the corresponding density.
13. Bring the volume of the cell suspension in each tube up to 2.2 ml with Medium iS.
○ If the volume in the tube exceeds 2.2 ml, centrifuge the required volume of cell suspension at 200 x g for 4
minutes, remove the supernatant, and resuspend the pellet in 2.2 ml Medium iS.
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514. Aspirate diluted iMatrix-511 silkfrom each newly coated well and add 1 ml cell suspension to each well.
○ Most of the diluted iMatrix-511 silk should be aspirated but not completely to prevent the coated well fromdrying before adding the cell suspension. The cell suspension should be added to the well immediately
after the diluted iMatrix-511 silk is removed. Handle one well after another.
15. Leave the plate flat at room temperature for 10 minutes.
16. Incubate the culture plate at 37°C, 5% CO2 overnight.

Day 1~8 hours

IMPORTANT! Observe all wells under a microscope and confirm that all 6 wells show 50-70% confluency fortransfections with QNS-mRNA-P. If there are any wells that do not fall within the range of confluence, do not use them.
First Treatment
1.

Thaw 1 vial of QNS-mRNA-P on ice for 30 minutes and warm Opti-MEM and Medium S at room temperature for
20-30 minutes.
2. Prepare QNS by the following steps:○ Tap the QNS-mRNA-P tube 3 times and then briefly spin it down before use.
○ Prepare a 15 ml tube and a 1.5 ml tube with 825 μl Opti-MEM each. Label the 15 ml tube “Mix 1” and the 1.5
ml tube “Mix 2”.
○ Add 16.5 μl Lipofectamine MessengerMax (LMM) to the Mix 1 tube and mix by brief vortexing. Leave it atroom temperature for 10 minutes (Mix 1). Keep the rest of LMM at 4°C for later treatments.
○ IMPORTANT! Immediately before 10 minutes pass (i.e., around 8 minutes), add the entire contents of the
QNG1-mRNA vial to the other 1.5 ml tube with Opti-MEM (Mix 2). Mix by tapping 5 times. Do not vortex.
○ 10 minutes after mixing LMM with Opti-MEM, add Mix 2 into Mix 1, and pipet up and down 8-10 times. This
mixture is called QNS. Leave QNS at room temperature for 5 minutes and no longer.
Mix 1 Reagents

VolumeMix 2 Reagents

VolumeOpti-MEM

825 µl

Opti-MEM

825 µl

LMM

16.5 µl

QNS-mRNA-P~33 µl3. Add 6.6 ml Medium S to QNS and pipet up and down 2-3 times to mix.
4. Working with up to 2 wells at a time, aspirate the old medium out and add 1.25 ml of QNS mixture to each well.
Repeat until QNS mixture has been added to all wells.
5. Incubate the culture plate at 37°C, 5% CO2 for 2.5 hours.
Second Treatment
1. Pipet out the medium from each well and add 1 ml Medium S.
2. Incubate the culture plate at 37°C, 5% CO2 for 2 hours.
○ Put 1 vial of QNS-mRNA-P on ice when the incubation above reaches 1.5 hours and leave it for 30 minutes.
3. Repeat Steps 2-5 of the previous “First Treatment” section.
Medium Change and Drug Selection
1. Warm Medium N(S1) at room temperature for 20-30 minutes.
2. Transfer 10 ml Medium N(S1) into a tube and add puromycin to it at the predetermined optimal concentration (seeearlier section on “Drug Selection”).
3. Pipet out the medium from each well and add 1.5 ml Medium N(S1) with puromycin.
4. Incubate the culture plate at 37°C, 5% CO2 overnight.

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6Day 2~8 hours

IMPORTANT! Observe the QNS-treated cultures to make sure that they are reaching confluency (≥90%). If the culturesare <50% confluent and show signs of cell death (e.g., many floating cells), users should skip the third and fourth
treatments on Day 2 and proceed directly to the steps described in Day 3. The protocol will then be accelerated by oneday.
Third Treatment
1.

Thaw 1 vial of QNS-mRNA-P on ice for 30 minutes and warm Opti-MEM and Medium N(S1) at room temperaturefor 20-30 minutes.
2. Repeat Steps 2-5 of the previous “First Treatment” section using Medium N(S1) in place of Medium S to prepare
QNS mixture.
Fourth Treatment
1. Pipet out the medium from each well and add 1 ml Medium N(S1).
2. Incubate the culture plate at 37°C, 5% CO2 for 2 hours.
○ Put 1 vial of QNS-mRNA-P on ice when the incubation above reaches 1.5 hours and leave it for 30 minutes.
3. Repeat Steps 2-5 of the previous “First Treatment” section using Medium N(S1) in place of Medium S to prepare
QNS mixture.
Medium Change and Drug Selection
1.

Transfer 10 ml Medium N(S1) into a tube and add puromycin to it at the predetermined optimal concentration (seeearlier section on “Drug Selection”).
○ If more than 90% of the cells show resistance to puromycin at the concentration used on Day 1, consider
increasing its concentration.
2. Pipet out the medium from each well and add 1.5 ml Medium N(S1) with puromycin.
3. Incubate the culture plate at 37°C, 5% CO2 overnight.

Day 3< 1 hourMedium Change
1. Warm Medium N(S1) at room temperature for 20-30 minutes.
2. Transfer 10 ml Medium N(S1) into a tube and add puromycin to it at the predetermined optimal concentration (seeearlier section on “Drug Selection”).
3. Pipet out the old medium from each well and add 1.5 ml Medium N(S1) with puromycin.
○ IMPORTANT! For medium change, a small volume of the old medium (i.e., just enough to cover thesurface of the well) should be left. The fresh medium should be applied along the wall of the well very slowly.
4. Incubate the culture plate at 37°C, 5% CO2 overnight.

Day 4

4-6 hours

New Plate Preparation
IMPORTANT! This kit can accommodate replating to all wells of either a 6-well, a 24-well, or a 96-well plate. Refer to thetables for the recommended volumes. Please note that the volumes are per well in Table A and per plate in Table B.
1.
2.
3.
4.
5.

Vortex ornithine briefly and centrifuge it at a maximum speed for 1 minute.
Add ornithine to each well of a new plate in the volume specified in Table A.
Incubate the plate at 37°C, 5% CO2 for at least 2 hours (or at 4°C overnight one day before plating).
Thaw laminin and chill specified amounts of PBS on ice for 20-30 minutes.
Add laminin to chilled PBS in the volume specified in Table B. Mix well.

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○All PBS washes should be done dropwise and with room temperature PBS. Chilled PBS is only for thecoating step.
6. Aspirate the supernatant from each well and add PBS in the volume specified in Table A.
7. Repeat Step 6.
8. Aspirate PBS from each well and add diluted laminin according to Table A.
9. Incubate the plate at 37°C, 5% CO2 for at least 2 hours.
Table A. Recommended volumes per well for different plate formats.
Recommended volume per well
Reagents

6-well plate24-well plate

96-well plate

Ornithine

1.5 ml300 µl

50 µl

PBS

2 ml

500 µl100 µlDiluted laminin

1.5 ml300 µl

50 µl

Medium N(S1P)

500 µl200 µl

35 µl

Table B. Recommended volumes per plate for different plate formats.
Recommended volume per plate
Reagents
Diluted laminin

Medium N(S1P)

6-well plate24-well plate

96-well plate

Laminin

100 µl

80 µl

53 µl

Chilled PBS

10 ml

8 ml

5.3 mlMedium N(S1)

38 ml

52 ml

42 ml

Component P

38 µl

52 µl

42 µl

Media Preparation
1.

While the plate is incubating, prepare Medium N(S1P) using the volumes indicated in Table B above.
○ Thaw Component P for 20-30 minutes at the temperature indicated in the “Contents” table on page 1.
○ Warm Medium N(S1) at room temperature for 20-30 minutes.
○ Tap the Component P tube(s) 3 times and then briefly spin down before use○ Store Medium N(S1P) for up to 2 weeks at 4°C.
○ Store the remaining Medium N(S1) and thawed Component P at 4°C.
2. After the laminin incubation, aspirate most, but not all, of the supernatant from each well and add PBS in theamount specified in Table A above. Add the PBS dropwise to each well.
3. Aspirate most, but not all of the PBS and add Medium N(S1P) in the volume specified in Table A above.
4. Incubate the plate at 37°C, 5% CO2 until cells are ready for plating.
Passaging Cells
IMPORTANT! For the following steps, gently pipet and add solutions. Differentiating cells are delicate and should be
handled with great care. Steps 2-9 below are critical. Perform these steps one well at a time. Refer to the images below tosuccessfully manage cell treatment and dissociation.

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1.
2.
3.
4.
5.
6.
7.
8.

Make sure that Solution D1 is at room temperature for at least 1 hour before use.
Pipet out the old medium from one well and add 1 ml PBS to the well.
Pipet out the PBS from the well and add 300 µl Solution D1.
Rock the plate 3 times to spread the Solution D1 evenly.
Incubate the cultures at 37°C, 5% CO2 for 3 minutes.
Carefully pipet out Solution D1 from the well and add 750 μl Medium N(S1P) along the wall of the well.
Tap each long side of the plate 5 times for a total of 10 times (no pipetting should be done).
Observe cells and/or cell aggregates floating in the well under a microscope. It is normal that 10-20% of cells
remain attached to the well bottom after pipetting. These clusters of cells are not supposed to be lifted. Do not
attempt to detach all of the cells remaining on the well bottom.
9. Collect 750 μl cell suspension from the well and transfer to a tube.
10. Repeat steps 2-9 for the rest of the wells.
11. Gently pipet the cell suspension up and down up to 5 times to break the cell aggregates. Excessive pipetting candamage the already-suspended neuronal cells.
12. Count cells and determine viability.
13. Prepare specified amounts of a 1 x 10⁶ live cells/ml cell suspension using Medium N(S1P) based on the table below.
14. Add cell suspension to the center of each well. Since each well already has Medium N(S1P), the total volume of themedium in each well is indicated in the table below.
Recommended Amounts
6-well plate24-well plate

96-well plate5 x 105 cells

1 x 105 cells

1.5 x 104 cells

Req vol of cell suspension (1 x 10⁶ viable cells/ml)● (Vol of cell suspension/well x # of wells) +10% extra

3.3 ml2.64 ml

1.6 mlVolume of cell suspension/well

500 µl100 µl

15 µl

Total volume/well● Medium N(S1P) + cell suspension

1 ml

300 µl

50 µl

Viable cells/well

15. Incubate the culture plate at 37°C, 5% CO2 overnight.

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9Day 5< 1 hourMedium Change
IMPORTANT! It is optional, but recommended, to include the PBS wash if cell death/floating cells are observed.
1. Warm Medium N(S1P) at room temperature for 20-30 minutes.
2. Pipet out the old medium from each well and * add Medium N(S1P) according to the table below.
*(Optional) Slowly add 1 ml PBS alongside the wall of each well to avoid lifting attached cells. Gently pipet out PBS
before adding Medium N(S1P).
Required volume per well
Reagents
Medium N(S1P)

6-well plate24-well plate

96-well plate

2 ml

800 µl150 µl3. Incubate the culture plate at 37°C, 5% CO2 for 2 days.

Days 7-9< 1 hourMedium Change
1. Warm Medium N(S1P) at room temperature for 20-30 minutes.
2. Pipet out half the old medium from each well and add Medium N(S1P) according to the table below.
Required volume per well
Reagents
Medium N(S1P)

6-well plate24-well plate

96-well plate

1 ml

400 µl75 µl

3. Incubate the culture plate at 37°C, 5% CO2.
4. Repeat steps 1-3 every 2 days.

Day 10
Assay or Continuous Maturation●●

Differentiated neurons can be observed on Day 5. For more mature neurons, we recommend culturing cells until
Day 10. From Day 10, users may maintain differentiated neurons in the medium best suited for their needs, though
we recommend Quick-Neuron™ Sensory Maintenance Medium, Catalog Number: SS-MM.
Differentiation into sensory neurons after using Quick-Neuron™ Sensory - mRNA Kit (Large) can be confirmed withthe markers SCN9A, PRPH, ISL1, and BRN3A.

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10Appendix

Figure 1. Representative phase contrast images of Quick-Neuron™ Sensory - mRNA Kit cell cultures on days 1, 2, 3, 4, 6, and 9
post-differentiation (scale bar = 100 μm).

Figure 2. Immunofluorescent staining of Quick-Neuron™ Sensory - mRNA Kit cell cultures shows typical neurite growth and expressionof PRPH, TUBB3, BRN3A, and ISL1 on day 10 post-differentiation (scale bar = 100 μm). Staining conditions: Peripherin Rabbit
polyclonal antibody (ThermoFisher, Catalog number: PA110018, 1:2000 dilution) was used in combination with a secondary antibody(Invitrogen, Catalog number: A32731 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488,
1:500 dilution). Anti-β-III tubulin monoclonal antibody (R&D Systems, Catalog Number: MAB1195, 1:250 dilution) in combination with asecondary antibody (Invitrogen, Catalog Number: A11032 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody,
Alexa Fluor 594, 1:500 dilution). BRN3A(14A6):mouse antibody (Santa Cruz Biotechnology, Catalog number:sc-8429 ,1:200 dilution) in
combination with a secondary antibody (Invitrogen, Catalog Number: A11032 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed
Secondary Antibody, Alexa Fluor 594, 1:500 dilution). ISL1 (DSHB, Catalog number: 39.4D5, 1:200 dilution) antibody in combination
with a secondary antibody (Invitrogen, Catalog Number: A11032 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary
Antibody, Alexa Fluor 594, 1:500 dilution). Nuclei were counterstained with Hoechst 33342.

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Figure 3. Real-time quantitative PCR analysis of expression levels of sensory neuron-associated genes SCN9A, PRPH, ISL1, and
BRN3A were examined. The graph shows a comparison of gene expression in Quick-Neuron™ - Sensory mRNA Culture on day 10 withgene expression in Human Dorsal Root Ganglion total RNA (TaKaRa, Catalog Number: 636150). The relative gene expression isnormalized to phosphoglycerate kinase 1 (PGK1), and then calculated as a fold induction relative to hiPSCs as a control. Error bars show
standard deviation. Primers used are listed in Table 1.
Table 1. List of PCR primers used in Figure 3
Gene

Forward primer

Reverse primer

Primer concentration

SCN9A

CCGTGGAACTGGCTGGATTTTTGTCTTCAGGCCTGGGATT

250 nM

PRPH

GCCTGGAACTAGAGCGCAAG

CCTCGCACGTTAGACTCTGG

250 nM

ISL1

TCTGTGGGCTGTTCACCAACTGTA

GCCGCAACCAACACATAGGGAAAT

250 nM

BRN3A

GCACACATTCACACAGTGGTAACAG

AGGCTCAGCAGACATACATGGACAG

250 nM

PGK1

GTATGCTGAGGCTGTCACTCG

CCTTCCAGGAGCTCCAAACTGG

250 nMelixirgensci.com

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