1. Total RNA Isolation from cultured cells.
1. Wash cultured cells 2X with 8 ml PBS (without calcium, without magnesium) at RT.(For
100 mm dish use 5 ml PBS)
2. Add 8 ml TRIzol reagent (Gibco/BRL) to cells in T-75 flask.
(For 100 mm dish use 5 ml TRIzol reagent.)
3.
Transfer lysed cell solution to a 15 ml flacon tube.
4. Incubate at RT for 5 minutes.
5. Add 1.6 ml Chloroform:Iso-Amyl alcohol (24:1)
to each tube.
6. Shake vigorously for 30 seconds.
7. Incubate at RT for 3 minutes.
8. Spin in centrifuge at 4oC for 15 minutes at 4000
RPM.
9. Transfer upper phase (clear solution only; no white goop) to a new 15 ml tube.
10. Add 4 ml of Isopropanol (2-propanol) to each
tube.
11. Mix by inverting several times.
12. Incubate at RT for 10 minutes.
13. Spin for 30 minutes at 4oC at 4000 RPM.
14. Remove supernatant
with a pipette very carefully.
15. Resuspend pelleted RNA using pipette in 1 ml of 70 % Ethanol.
16. Transfer to a microfuge tube.
17.
Spin for 10 minutes at 4oC at 14,000 RPM.
18. Remove supernatant with a pipette.
19. Aspirate remaining drops.
20. Air-dry pellet for
5-10 minutes at RT.
21. Resuspend RNA pellet in 80 ul DEPC treated water.
22. Spec 2 ul in 198 ul of water.
23. Store RNA at -80oC.
Jeffrey
D. Randall, Takumi Yoshida, Ryoji Kojima, Steven Gullans © Page 2 9/6/2001
Jeffrey D. Randall, Takumi Yoshida, Ryoji Kojima, Steven
Gullans © Page 2 9/6/2001
Microarray Protocols
Basic knowledge of microarray.
Basic knowledge of microarray 2.
MicroArray Procedure
(Modified by Ryoji Kojima, Takumi Yoshida and Jeffrey Randall,
8.20.2000)
1. Total RNA Isolation from cultured cells.
2. DNase Treatment of Total RNA
3. Making the single strand
cDNA probe.
4. Automated Slide Processor (ASP) Version for hybridization.
5. Washing microarrays in ASP.
7. Pre-hybridization of the processed slides (NON-Automated version).
8. Hybridization of Cy3 + Cy5 probe
to glass array (NON-Automated version).
9. Preparation of Dendrimer Cy3 and Cy5.
10. Washing unbound probe from glass array
(NON-Automated version).
11. Hybridization of Dendrimers (Cy3 and Cy5) to Array (NON-Automated version).
12.Washing unbound
dendrimer from glass array(NON-Automated version).
Other Microarray Protocols (1)
Other Microarray Protocols (2)
Web Guider
Ch 8.Immunohistoch / immunology
Ch 10.GC/MS, NMR and Proteomics