Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA

Article Details

Protocols » Article Details

Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA

Date Added: March 02, 2008 09:00:14 PM

Author:

Category: DNA Microarray Protocols

Background on the Protocol - Preparation of Fluorescent DNA Probes from Human mRNA

DNA microarrays are ordered arrays of DNA molecules complementary to the genes of interest. They are spotted on the array by robotic equipment using a glass slide substrate. The expression of genes in cells can then be assessed on 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.

Step 1 - Microarray Probe Preparation Method

Label two microcentrifuge tubes Cy3 and Cy5, respectively.
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.
Heat to 65°C for 10 min and cool on ice.
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 dNTP, 1 mM Cy3 or Cy5, 2.0 μl of Superscript II
Incubate at 42°C for 1 hr.
Add 1 μl of SSII (reverse transcriptase booster) to each sample.
Incubate for an additional 0.5 to 1.0 hr.
Add 15 μl of 0.1 M NaOH to degrade the RNA.
Incubate at 70°C for 10 min.
Add 15 μl of 0.1 M HCl to neutralize the reaction.
Adjust the volume to 400 μl with TE.
Combine both probes into 1 Centricon-30™ Filter Unit.
Wash #1: Centrifuge at 14 krpm in a Centricon-30™ micro-concentrator for 7 min (see Hint #1).
Wash #2: Add 45 μl of TE and centrifuge again at 14 krpm in a Centricon-30™ micro-concentrator for 7 min.
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 and Centrifuge at 14k rpm in a Centricon-30™ micro-concentrator for 7 min (see Hint #2).
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.
Invert the Centricon-30™ into a fresh tube and centrifuge briefly at 14 krpm to recover the probe.

Buffers for Microarray Probe Preparation

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)

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

Protocol Microarray Probe Labeling Tips & 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.

Ratings You must be logged in to leave a rating.
Average rating: (0 votes)

Comments