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RNA extraction from Spheroids
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Introduction

It is recognized that spheroid gene expression pattern is different from that of monolayer cultured cell. To know the gene expression profile, RNA extraction step is needed.
Here, we introduce RNA extraction method from spheroid cultured in NanoCulture Dish.

Materials, reagents and equipment

1. Cell
HT-29(human colorectal cancer cell) ATCC ®HTB-38™
2. Reagents
Media McCoy’s 5a supplemented 10%FBS and antibiotics
(Heat-inactivated FBS must be used)
QIAshredder QIAGEN Cat# 79654 or 79656
RNeasy Plus Mini Kit QIAGEN Cat# 74134 or 74136
3. Materials
NanoCulture Dish MH pattern,
Low-Binding, 35 mm
: ORGANOGENIX Cat# NCD-LH35

Methods

【3D Cell Culture】

1. Add media (2 mL / dish) in Nano Culture Dish (NCD)
2. Remove bubbles from the edge of the dish bottom by pipetting.
3. Stand for 15 – 20 minutes, at room temperature or inside the CO2 incubator to remove micro-bubbles.
4. Discard 1 mL medium from the dish.
5. Prepare HT-29 cell suspension (1×105cells/mL)
6. Add HT-29 cell suspension (2×105cells/2 mL/dish) in NCD. So total media volume will be 3ml in NCD.Please try not to scratch well bottom which may disturb cell spheroid growth. It may results monolayer cell growing.
7. Stand for 20 – 30 minutes at ambient condition after seeding cells for better cell attachment on the dish.If NCP plate place inside the CO2 incubator before the cells attach to NCP, it may result in an uneven distribution of spheroids due to vibration and convection micro-currents while moving the plates.
8. Set an accessory insert-grid in the dish.
9. Incubate for appropriate time at 5 % CO2, 37℃.

【Extraction total RNA】

10. Remove an insert-grid and collect spheroids with media into a collection tube by pipetting.
11. Wash the dish with 1 mL medium and add into the collection tube.(Total 4 mL spheroid suspension)
12. centrifuge (4℃、300 xg), and discard the sup.
13. Extract total RNA using QIAshredder and RNeasy Plus Mini Kit. (according to the procedure)
14. Extracted RNA quality should be checked by the following criteria.
Yield (RNA concentration)
Purity(A260/A280 ratio)
Integrity (28S/18S rRNA ratio)

Example of Results

【Target of RNA yields on several culturewares】

35 mm dish 24-well plate 96-well plate 384-well plate
Cell number
(per dish or well)
200,000 60,000 10,000 3,000
RNA yields
(after 7 days culture)
2-6
µg/dish
0.6-1.8
µg/well
0.1-0.3
µg/well
0.03-0.1
µg/well