Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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The material on this page is part of Chapter 10, which is shown in full as a preview on this site.

Chapter 10: Nucleic Acid Platform Technologies

Rando Oliver, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

INTRODUCTION

Direct Cyanine-dUTP Labeling of RNA

(Protocol summary only for purposes of this preview site)

This is the simplest method to label RNA for use in expression analysis. RNA is reverse-transcribed using both oligo(dT) and random hexamers as primers. The random hexamers improve overall efficiency of labeling, especially at the 5 end of the RNA. Fluorescently labeled dUTP is incorporated into the cDNA. After reverse transcription, the RNA is degraded, and the labeled cDNA is purified from unincorporated Cy dyes. Finally, samples labeled with Cy3 and Cy5 dyes are mixed and combined with blocking nucleotides and used for hybridization, as described in Protocol 10.


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 Protocol 5: Direct Cyanine-dUTP Labeling of RNA

This is the simplest method to label RNA for use in expression analysis. RNA is reverse-transcribed using both oligo(dT) and random hexamers as primers. The random hexamers improve overall efficiency of labeling, especially at the 5 end of the RNA. Fluorescently labeled dUTP is incorporated into the cDNA. After reverse transcription, the RNA is degraded, and the labeled cDNA is purified from unincorporated Cy dyes. Finally, samples labeled with Cy3 and Cy5 dyes are mixed and combined with blocking nucleotides and used for hybridization, as described in Protocol 10.


MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

Recipes for reagents specific to this protocol, marked <R>, are provided at the end of the protocol. See Appendix 1 for recipes for commonly used stock solutions, buffers, and reagents, marked <A>. Dilute stock solutions to the appropriate concentrations.

Reagents

  • Cy3-dUTP or Cy5-dUTP (GE Healthcare Life Sciences)
  • DEPC-treated H2O
  • HCl (0.1 N)
  • NaOH (0.1 N)
  • Oligo(dT) (2 g/L)
  • Random hexamers (4 g/L)
    • N6 random hexamer can be ordered from any oligonucleotide company and made up at 5 mg/mL in RNase-free TE or water.
  • Reverse transcriptase, 200 U/L (SuperScript II; Life Technologies)
    • First strand buffer <R> and DTT, both required for preparation of the master reagent mix in Step 3, are provided with SuperScript II.
  • Total RNA (from Protocol 3 or 4)
  • Unlabeled dNTPs (low dTTP) stock <R>

Equipment

  • Heat block set at 65C
  • MinElute kit (QIAGEN, catalog no. 28004)
  • Thermal cycler


METHOD
RT Reaction

  • 1. Prepare the following RNA/oligo reaction mixtures in separate microcentrifuge tubes.
  • 2. Heat the tubes for 10 min to 65C, and cool on ice to anneal the primers to the RNA.
  • 3. While the RNA/oligo reaction mixtures are incubating, prepare two master reaction mixtures, one containing Cy3-dUTP and one with Cy5-dUTP, in a volume sufficient to transfer 14.6 L of each to an RNA/oligo reaction mixture.
  • 4. To the Cy3 RNA/oligo reaction mixture, add 14.6 L of the Cy3 master reaction mixture (Step 3) to each tube. To the Cy5 RNA/oligo reaction mixture, add 14.6 L of the Cy5 master reaction mixture (Step 3) to each tube. Incubate for 1 h at 42C.
  • 5. Add 1 L of SuperScript II enzyme (200 U/L) to each reaction and thoroughly mix the reaction components with a pipette. Incubate for an additional 1 h.
  • 6. Degrade the RNA by adding 15 L of 0.1 N NaOH to each reaction and incubating for 10 min at 65C70C.
  • 7. Neutralize each reaction by adding 15 L of 0.1 N HCl.

Cleanup

If you want to determine the amount of fluorophore incorporated, then clean up the Cy5- and Cy3-labeled cDNA samples on separate MinElute columns. However, if thin coverslips and a small probe volume will be used during the hybridization (Protocol 10), it may be necessary to purify the Cy5- and Cy3-labeled cDNA samples together or use vacuum centrifugation to reduce the volume after elution.

  • 8. Add 600 L of Buffer PB (binding buffer) to each sample.
  • 9. Assemble a MinElute column on a 2-mL collection tube.
  • 10. Add the entire 660 L or 720 L (volume depends on whether each color sample is being cleaned up separately or together) to a MinElute column. Centrifuge the column for 1 min at 10,000g. Discard the flowthrough, and reuse the 2-mL tube.
  • 11. Add 750 L of Wash buffer PE to the column. Centrifuge for 1 min at 10,000g. Discard the flowthrough, and reuse the 2-mL tube.
  • 12. Centrifuge again at maximum speed for 1 min to remove residual ethanol.
  • 13. Place the column in a fresh 1.5-mL tube. Add 10 L of H2O to elute. Allow the Elution buffer to stand for at least 2 min.
  • 14. Centrifuge at maximum speed for 1 min. Add 10 L of H2O to elute. Allow the Elution buffer to stand for at least 2 min before spinning.
  • 15. Centrifuge at maximum speed for 1 min.
  • 16. Measure the volume of the eluate for each sample. The volume should be 18 L for each column.


DISCUSSION

The output of this protocol will be Cy5-labeled cDNA and Cy3-labeled cDNA. The DNA can be used immediately to hybridize to a microarray, or it can be stored in foil, to prevent bleaching, at 4C for up to a week. A useful spot check for labeling success is the color of the labeled material after unincorporated nucleotides have been removed (see Step 16). A good labeling will result in blue Cy5-DNA and red Cy3-DNA.


RECIPES

It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

First-Strand Buffer
Unlabeled dNTPs (Low dTTP) Stock

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