RNA Extraction with TRIZOL

Wear gloves when handling trizol, work RNase-free!

1. Aspirate medium from cells grown in dishes
2. Wash with 1XPBS
3. In the fume hood, add 1 ml TRIzol per ~1x106 cells, that is when culture flask(25 cm2)reaches confluency; or 4ml of Trizol to cells on plastic of 10cm plate;2.5 ml on 60 mm plate;1 ml on 35mm plate.
4. Pipette up and down for 2-3 min until solution is no longer viscous;
5. Centrifuge for 10 min at 9000 rpm at C( No dissolved protein will be pelleted),and transfer supernatant into fresh tube
6. Add 20% of Chloroform for Trizol ( i.e. 0.2 ml Chloroform for each 1 ml Trizol )
7. Close tubes firmly and vortex vigorously for at least 15 seconds(UP TO 1 min)
8. Let sit at room temperature for 2min 30sec
9. Centrifuge for 15 min at 9000rpm at 4C
10. Transfer upper, aqueous phase to fresh 1.5ml tube(0.5ml/tube), avoid transfer of ANY interface!!
11. Add equal volume isopropanol to the clean supernatant and incubate at RT for 15 min.
12.Centrifuge at 15,000 rpm for 15 minutes to pellet the RNA.
13.Discard the supernatant and resuspend the pellet in 70% ethanol. The RNA can be stored in 70% ethanol at -20°C until use.
14.Prior to use, centrifuge at 15,000 rpm for 15 minutes at 4°C and discard supernatant.
15.Remove the supernatant, and air dry the RNA pellet for 5 min at RT
16.Resuspend the pellet in diethylpyrocarbonate (DEPC) treated water 30ul or RNase-free TE buffer forlabeling.

tissue culture dish vs growth area

pc12 information

Cell Biology

ATCC® Number: CRL-1721™ Price: $256.00
Additional information about this cell line
Designations: PC-12 Depositors: B Patterson
Biosafety Level: 1 Shipped: frozen
Medium & Serum: See Propagation Growth Properties: loosely adherent, multicell aggregates
Organism: Rattus norvegicus (rat) Morphology: polygonal


Source: Organ: adrenal gland
Disease: pheochromocytoma
Cellular Products: catecholamines; dopamine; norepinephrine [1163]
Permits/Forms: In addition to the MTA mentioned above, other ATCC and/or regulatory permits may be required for the transfer of this ATCC material. Anyone purchasing ATCC material is ultimately responsible for obtaining the permits. Please click here for information regarding the specific requirements for shipment to your location.

Related Cell Culture Products
Applications: transfection host (Roche FuGENE® Transfection Reagents
technology from amaxa)
Receptors: nerve growth factor (NGF), expressed
Tumorigenic: YES
Cytogenetic Analysis: 40 chromosomes; 38 autosomes plus XY [1163]
Gender: male
Comments: The PC-12 cell line was derived from a transplantable rat pheochromocytoma. [1163]
The cells respond reversibly to NGF by induction of the neuronal phenotype. [1163]
The cells do not synthesize epinephrine. [1163]

Propagation: ATCC complete growth medium: The base medium for this cell line is ATCC-formulated F-12K Medium, Catalog No. 30-2004. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 2.5%; horse serum to a final concentration of 15%.

Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37.0°C

Subculturing: Protocol: Volumes used for this protocol are for a 75cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.1. Remove and discard old culture medium.2. Pipet 10 ml fresh medium over the cell sheet and scrape.3. Aspirate cells with a small bore pipette to break up clusters.4. Add appropriate aliquots of the cell suspension to new 75 cm2 flask with 15 ml fresh growth medium. Seed flask at 1.0 x 10(4) to 3.0x 10(4) viable cells / cm2.Or use subcultivation ratio of 1:3 twice weekly Subculture when cell density reaches between 1.0x 1 0(5) to 2.0x 10(5) viable cells / cm2.5. Place culture vessels in incubator at 37ºC.PC-12 cells adhere poorly to plastic and tend to grow in small patches of loosely attached cells.Attachment can be enhanced by coating the flasks with Bovine Collagen I or using Corning® CellBIND® Surface Flasks (Free Samples)

Subcultivation Ratio: 1:3 twice weekly
Medium Renewal: Every 2 to 3 days
Preservation: Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO

Storage temperature: liquid nitrogen vapor phase
Doubling Time: 48 hrs
Related Products: Recommended medium (without the additional supplements or serum described under ATCC Medium):ATCC 30-2004
recommended serum:ATCC 30-2020
recommended serum:ATCC 30-2040
References: 1162: Levi A, et al. Molecular cloning of a gene sequence regulated by nerve growth factor. Science 229: 393-395, 1985. PubMed: 3839317
1163: Greene LA, Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73: 2424-2428, 1976. PubMed: 1065897
22344: Biocca S, et al. A macromolecular structure favouring microtubule assembly in NGF- differentiated pheochromocytoma cells (PC12). EMBO J. 2: 643-648, 1983. PubMed: 6641712
33014: Weber E, et al. Distinct functional properties of Rab3A and Rab3B in PC12 neuroendocrine cells. J. Biol. Chem. 271: 6963-6971, 1996. PubMed: 8636125