In an autoclaved PCR tube Add: In an autoclaved PCR tube Add:
Cy3 (Red) Cy5 (Green)
Total
RNA 10 ug Total RNA 10 ug
RT primer (Cy3,1pmole/ul) 1.0 ul RT primer (Cy5, 1pmole/ul) 1.0 ul
DEPC treated water x ul DEPC Treated water
x ul
Total reaction volume 10 ul Total reaction volume 10 ul
Heat in PCR machine at 80oC for 10 minutes.
Chill to 42oC and hold.
Use new
PCR caps as heat denatures plastic and allows evaporation!
In a separate microfuge tube make the reaction buffer (# of samples + 1).
Single
channel Double channel (Cy3 and Cy5)
5x RT Buffer (Gibco/BRL) 4 ul 8 ul
0.1 M DTT (Gibco/BRL) 2 ul 4 ul
10 mM dNTP’s 1 ul 2 ul
RNase Inhibitor
(40 U/ul, Promega) 1 ul 1 ul
DEPC treated water 1 ul 2 ul
Superscript II (200 U/ul, Gibco/BRL) 1 ul 2 ul
Total reaction volume 10 ul
20 ul
Add 10 ul of Reaction Buffer to each PCR tube.
Mix well with pipette and incubate at 42oC in PCR machine for 2 hours.
Use new PCR
caps to prevent evaporation!
Add 3.5 ul of 0.5 M NaOH/50 mM EDTA to each tube.
Incubate at 65oC for 10 minutes to denature DNA/RNA hybrids.
Add:
5
ul of 1 M Tris/HCl, pH 7.5 to neutralize the reaction mixture.
70 ul of DEPC treated water (to dilute reaction and bring volume to
100 ul)
Combine all the Cy3 and Cy5 RT reaction mixtures in a microfuge tube.
Add:
0.1 volumes (20 ul) of 3 M NaOAc
2.5 volumes (550 ul)
of 100% ethanol (-20oC)
Mix by inversion several times.
Place in DRY ICE/Ethanol bath for 30 minutes (label with tape as marker will
run)
or place in –80oC for 1 hour.
Spin at 14,000 RPM for 20 minutes at Room Temp.
Remove supernatant with pipette carefully.
Add 500
ul of 70% Ethanol (-20oC)
Spin down at 14,000 for 15 minutes at Room temp.
Remove the supernatant using a pipette, carefully.
Air-Dry
the pellet for 2 - 5 minutes at RT.
Jeffrey D. Randall, Takumi Yoshida, Ryoji Kojima, Steven Gullans © Page 4 9/6/2001
Jeffrey D. Randall,
Takumi Yoshida, Ryoji Kojima, Steven Gullans © Page 4 9/6/2001
Re-suspend the pellet in 15 µl Dendrimer hybridization buffer (pre-warmed
to 65oC).
(Tube #6 in Genisphere kit, 0.25 M NaPO4, 4.5% SDS, 1 mM EDTA, 1X SSC).
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