I cannot properly isolate sticky cell lines for extraction of total RNA. I am getting low concentration of RNA after extraction (poor band on agarose gel).
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W.M. Keck Foundation Biotechnology Microarryay Resource Laboratory at Yale University
TRIZOL RNA Isolation Protocol
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Please do not make copies of or distribute this protocol.
A. Required reagents:
DEPC-treated water (Ambion)
TRIzol Reagent (Invitrogen)
Ice cold PBS
Cell scraper
70% ethanol
Isopropyl alcohol
B. Equipment and supplies:
Refrigerated centrifuge
Microcentrifuge
Micropipettors
Aerosol-barrier tips
Vortex mixer
Powder-free gloves
Centrifuge tubes
C. Safety:
Always use gloves and eye protection. Avoid contact with skin or clothing. Use in a
chemical hood. Avoid breathing vapor.
D: Homogenization:
1. Tissues:
Homogenize tissue samples in 1 ml of TRIZOL reagent per 50 to 100 mg of tissue using a
glass-Teflon or power homogenizer (e.g. Polytron, Tekmar’s TISSUEMIZER). The sample
volume should not exceed 10% of the volume of TRIZOL Reagent used for the
homogenization.
2. Cells grown in Monolayer:
Rinse cell monolayer with ice cold PBS once. Lyse cells directly in a culture dish by
adding 1 ml of TRIZOL Reagent per 3.5 cm diameter dish and scraping with cell scraper. Pass the cell lysate several times through a pipette. Vortex thoroughly. The
amount of TRIZOL reagent added is based on the area of the culture dish (1 ml per 10
cm2
) and not on the number of cells present. An insufficient amount of TRIZOL
Reagent may result in DNA contamination of the isolated RNA.
3. Cells Grown in suspension:
Spin cells for 5 min at 300 X g. Remove media and resuspend cells in ice cold PBS.
Pellet cells by spinning at 300 X g for 5 min. Lyse cells with TRIZOL Reagent by repetitive
pipetting or by passing through syringe and needle. Use 1 ml of the reagent per 5-10 X
106
of animal cells.
4. Incubate the homogenized sample for 5 minutes at room temperature to permit
the complete dissociation of nucleoprotein complexes. Centrifuge to remove cell
debris. Transfer the supernatant to new tube.
E. PHASE SEPERATION:
Add 0.2 ml of chloroform per 1 ml of TRIZOL Reagent. Cap sample tubes securely.
Vortex samples vigorously for 15 seconds and incubate them at room temperature for
2 to 3 minutes. Centrifuge the samples at no more than 12,000 x g for 15 minutes at 2
to 80C. Following centrifugation, the mixture separates into lower red, phenolchloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains
exclusively in the aqueous phase. Transfer upper aqueous phase carefully without
disturbing the interphase into fresh tube. Measure the volume of the aqueous phase
(The volume of the aqueous phase is about 60% of the volume of TRIZOL Reagent used
for homogenization).
F. RNA PRECIPITATION:
Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5
ml of isopropyl alcohol per 1 ml of TRIZOL Reagent used for the initial homogenization.
Incubate samples at 15 to 30oC for 10 minutes and centrifuge at not more than 12,000
x g for 10 minutes at 2 to 4oC. The RNA precipitate, often invisible before
centrifugation, forms a gel-like pellet on the side and bottom of the tube.
G: RNA WASH:
Remove the supernatant completely. Wash the RNA pellet once with 75% ethanol,
adding at least 1 ml of 75% ethanol per 1 ml of TRIZOL Reagent used for the initial
homogenization. Mix the samples by vortexing and centrifuge at no more than 7,500 x
g for 5 minutes at 2 to 8 oC. Repeat above washing procedure once. Remove all
leftover ethanol.
H. REDISSOLVING RNA:
Air-dry or vacuum dry RNA pellet for 5-10 minutes. Do not dry the RNA pellet by
centrifuge under vacuum. It is important not to let the RNA pellet dry completely as
this will greatly decrease its solubility. Partially dissolved RNA samples have an A260/A280
ratio < 1.6. Dissolve RNA in DEPC-treated water by passing solution a few times
through a pipette tip. I. SPECTROPHOTOMETRIC ANALYSIS:
Dilute 1 μl of RNA with 39 μl of DEPC-treated water (1:40 dilution). Using 10 μl
microcuvette, take OD at 260 nm and 280 nm to determine sample concentration
and purity. The A260/A280 ratio should be above 1.6. Apply the convention that 1 OD
at 260 equals 40 µg /ml RNA.
Important: For microarray experiments, total RNA isolated by TRIZOL method needs to
be further cleaned using RNeasy cleanup protocol.
REFERENCES:
Chomczynski P, Mackey K. (1995) Short technical report. Modification of the TRIZOL reagent
procedure for isolation of RNA from Polysaccharide-and proteoglycan-rich sources.
Biotechniques 19(6): 942-5.
TRIZOL Reagent technical insert (Invitrogen).
http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/RNA%20and%20mRNA/UM_TotalRNA.pdf
Hi, Ali Esmalei
Thanks very much for the Details, but I have no problem in RNA extraction from tissues, my problem is RNA extraction from sticky cell lines.
Hi, Ali Esmalei
excuse me , I make a mistake, Not only did you answer my questions, you even gave me accurate, detailed information, thanks a lot, good luck
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