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Malaysia University |
MICROARRAY: PREPARATION OF FLUORESCENT DNA PROBES FROM HUMAN mRNA
Microarray: Preparation of Fluorescent DNA Probes from Human mRNA
Overview
This protocol describes the production of probes labeled with the fluorescent dyes, Cy3 and Cy5, following the synthesis of cDNA from human mRNA and the hybridization of the probes to DNA microarrays. DNA microarrays are an ordered arrangement of DNA molecules complementary to the genes of interest that are "spotted" by robotic equipment onto a glass slide substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of the cells of interest and measuring the hybridization of the prepared cDNA to the spotted DNA of the microarray. The intensity of the fluorescence of the hybridized probes indicates the abundance of specific transcripts and thus reflects the expression levels of specific genes.
Procedure
A. Probe Preparation
1. Label two microcentrifuge tubes Cy3 and Cy5, respectively.
2. Combine the following in each tube to anneal the primer:
2 μg of mRNA
4 μg of a regular or anchored oligo-dT primer
Add ddH2O to a total volume of 10 μl.
3. Heat to 65°C for 10 min and cool on ice.
4. Add 10 μl of the following reaction mix to each Cy3 and Cy5 reaction:
6.0 μl of 5X First Strand Buffer
3.0 μl of 0.1 M DTT
0.6 μl of unlabeled dNTPs
1 mM Cy3 or Cy5
2.0 μl of Superscript II
5. Incubate at 42°C for 1 hr.
6. Add 1 μl of SSII (reverse transcriptase booster) to each sample.
7. Incubate for an additional 0.5 to 1.0 hr.
8. Add 15 μl of 0.1 M NaOH to degrade the RNA.
9. Incubate at 70°C for 10 min.
10. Add 15 μl of 0.1 M HCl to neutralize the reaction.
11. Adjust the volume to 400 μl with TE.
12. Combine both probes into 1 Centricon-30™ Filter Unit.
13. Wash #1: Centrifuge at 14 krpm in a Centricon-30™ micro-concentrator for 7 min (see Hint #1).
14. Wash #2: Add 45 μl of TE and centrifuge again at 14 krpm in a Centricon-30™ micro-concentrator for 7 min.
15. Wash #3: Add the following:
450 μl of TE
20 μg of Cot 1 human DNA
2 μl of 10 μg/ μl of polyA RNA
2 μl of tRNA
Centrifuge at 14 krpm in a Centricon-30™ micro-concentrator for 7 min (see Hint #2).
16. Concentrate to a volume of less than 10 μl. This volume is attained when the center of the membrane is dry and the probe forms a ring of liquid at the edges of the membrane.
17. Invert the Centricon-30™ into a fresh tube and centrifuge briefly at 14 krpm to recover the probe.
B. For 22 x 40 mm arrays
1. Adjust the volume to 20 μl with TE (see Hint #3).
2. Add the following for the final probe preparation (see Hint #4):
4.25 μl of 20X SSC
0.75 μl of 10% SDS (see Hint #5)
3. Denature the probe by heating for 2 min at 100°C.
4. Incubate at room temperature for 15 to 20 min.
5. Centrifuge at 14 krpm for 10 min.
6. Place 24 μl of the probe on the array underneath a 22 mm x 40 mm glass cover slip (see Hint #6).
7. Prepare a slide chamber with the humidity maintained by a small reservoir of 3X SSC by spotting approximately 3 to 6 μl of 3X SSC at each corner of the slide (as far away from the array as possible).
8. Hybridize at 65°c for 14 to 18 hr in the slide chamber.
9. Set up the 200 ml of washes in 250 ml chambers as follows:
Wash I: 2X SSC with 0.1% SDS
Wash II: 1X SSC (prepare two chambers)
Wash III: 0.2X SSC (see Hint #7)
10. Blot-dry the exterior of the chamber with towels and aspirate any remaining liquid from the water bath. Aspirate the space between the two chamber halves.
11. Unscrew the chamber and aspirate within the holes to remove the last traces of water-bath liquid.
12. Place the arrays, singly, in a rack, inside the Wash I chamber (a maximum of four arrays at a time).
13. Allow the cover slip to fall, or carefully use forceps to aid the removal of the cover slip if it remains stuck to the array. DO NOT AGITATE until the cover slip is safely removed. Then agitate for approximately 15 sec.
14. Remove the array with the forceps. Rinse in a Wash II chamber (without a rack).
15. Transfer to a Wash II chamber containing a rack (see Hint #8).
16. Wash the arrays by submersion and agitation for 30 sec in the Wash II chamber, then transfer the entire slide rack to the Wash III chamber.
17. "Spin-dry" by centrifugation in a slide rack in a Beckman GS-6 tabletop centrifuge at 600 rpm for 2 min.
18. Scan the arrays immediately.
Solutions
1 mM Cy3 or Cy5
(Amersham)
0.1 M DTT
Anchored oligo dT
4 μg/μl oligo dT
5'-TTT TTT TTT TTT TTT TTT TTV N-3'
Oligo-dT
4 μg/μl oligo dT
SSII
Reverse Transcriptase Booster
20X SSC
3.0 M NaCl
300 mM Sodium Citrate (pH 8.0)
0.75 μl of 10% SDS
TE
10mM Tris
1mM EDTA
tRNA
(Gibco-BRL, #15401-011)
10 μg/μl tRNA
PolyA RNA
10 μg/μl polyA RNA
(Sigma, #P9403)
5X First-Strand Buffer
375 mM KCl
250 mM Tris-HCl (pH 8.3)
15mM MgCl2
Cot1 human DNA
(Gibco-BRL)
Unlabeled dNTPs
10 mM dTTP
25 mM dGTP
25 mM dATP
25 mM dCTP
Superscript II
200 Units/μl Superscript II (Gibco-BRL)
BioReagents and Chemicals
dCTP
dTTP
dGTP
dATP
Magnesium Chloride
Tris-HCl
Cy5
Cy3
DTT
Tris
Potassium Chloride
Sodium Chloride
SDS
EDTA
Sodium Citrate
RNA, PolyA
tRNA
DNA, Cot1 human
Superscript II
oligo dT, anchored
Oligo-dT
Protocol Hints
1. Do not combine the probes until Wash 2 if re-purification of the Cy dye flow-through is desired.
2. The 'colored probe' will be visible in the centricon unit when concentrated.
3. Use 12 μl for 22 mm x 22 mm arrays.
4. Use 2.55 μl of 20X SSC and 0.45 μl of 10% SDS for 22 mm x 22 mm arrays.
5. When adding the SDS, wipe the pipette tip with clean, gloved fingers to remove any excess SDS.
6. Use 12 μl for a 22 mm x 22 mm cover slip.
7. The Wash I chamber and one of the Wash II chambers should each have a slide rack ready.
8. This step minimizes the transfer of SDS from Wash I to Wash II.
Citation and/or Web Resources
1.Lab website
