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
Indirect Aminoallyl-dUTP Labeling of RNA
(Protocol summary only for purposes of this preview site)This protocol is slightly longer than the simpler direct-labeling protocol, but it is significantly cheaper because of the high cost of Cy-dNTPs used in Protocol 5. This labeling procedure is called indirect because the fluorescent moiety is not incorporated during the reverse transcription reaction. Instead, a reactive nucleotide analog (aminoallyl-dUTP) is incorporated during reverse transcription, the cDNA is isolated, and then the cyanine dyes are incorporated by binding with the aminoallyl group to produce the desired fluorescent cDNA.
Protocol 6: Indirect Aminoallyl-dUTP Labeling of RNAThis protocol is slightly longer than the simpler direct-labeling protocol, but it is significantly cheaper because of the high cost of Cy-dNTPs used in Protocol 5. This labeling procedure is called indirect because the fluorescent moiety is not incorporated during the reverse transcription reaction. Instead, a reactive nucleotide analog (aminoallyl-dUTP) is incorporated during reverse transcription, the cDNA is isolated, and then the cyanine dyes are incorporated by binding with the aminoallyl group to produce the desired fluorescent cDNA.
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.
- aa-dUTP mixture (50) <R>
- Anchored oligo(dT) (Life Technologies)
- Anchored oligo(dT) primer is a mix of 12 primers, each having a sequence of 20 dT residues followed by two nucleotides VN, where V is dA, dC, or dG and N is dA, dC, dG, or dT. Thus, the VN anchor restricts annealing of the primer to the 5 end of the poly(A) tail of mRNA.
- Cyanine dyes (typically Cy5 and Cy3) (GE Healthcare Life Sciences)
- Dye comes dried and should be resuspended in 11 L of DMSO. This amount of material is sufficient for three labeling reactions. If the entire amount will not be used, prepare 3-L aliquots, dry down in a rotary vacuum, and store them at 20C.
- DTT (0.1 M)
- EDTA (0.5 M)
- HEPES (1 M, pH 7.5)
- NaHCO3 (50 mM, pH 9.0)
- NaOH (1 N)
- Reverse transcriptase (SuperScript II; Life Technologies)
- Reverse transcriptase buffer (5) (Life Technologies)
- RNasin (ribonuclease inhibitor)
- Total RNA (from Protocol 3 or 4)
- Heat block set at 67C, 70C, and 95C
- Lightproof box
- MinElute kit (QIAGEN, catalog no. 28204)
- Thermal cycler (42C)
- Zymo column (Zymo Research, catalog no. D3024)
- 1. Combine 30 g of total RNA and water to a final volume of 14.5 L. Add 1 L of 5 g/L anchored oligo(dT). Mix by pipetting. Heat for 10 min at 70C.
- 2. Cool the tube on ice for 10 min.
- 3. Pulse-centrifuge to bring the condensate to the bottom of the tube.
- 4. Prepare the Master mix just before use (be sure to add the enzymes last):
- 5. Add 14.5 L of Master mix to the tube of RNA. Pipette to mix. Incubate the reaction for 2 h at 42C.
- 6. Incubate the tube for 5 min at 95C. Transfer immediately to ice.
- 7. Add 13 L of 1 N NaOH and 1 L of 0.5 M EDTA to hydrolyze the RNA. Mix the reagents and pulse-centrifuge. Incubate the tube for 15 min at 67C.
- 8. Neutralize the reaction with 50 L of 1 M HEPES (pH 7.5). Vortex the tube and pulse-centrifuge.
-
9.
Purify the reaction over a Zymo column to remove unincorporated nucleotides.
- i. Add 1 mL of Binding buffer to the reaction. Mix by pipetting. Load half of the material onto the column. Centrifuge for 10 sec at maximum speed. Discard the flowthrough.
- ii. Add the remainder of the reaction. Centrifuge for 10 sec at maximum speed. Discard the flowthrough.
-
iii.
Add 200 L of Wash buffer to the column. Centrifuge for 30 sec at maximum speed.
- Typically, multiple individual samples will be labeled simultaneously, with at least two reactions for a given two-color microarray.
- iv. Add another 200 L of Wash buffer to the column. Centrifuge for 1 min at maximum speed. Discard the flowthrough, and centrifuge for 1 min at maximum speed.
- v. Add 10 L of 50 mM NaHCO3 (pH 9.0) to the filter. Incubate for 5 min at room temperature. Centrifuge for 30 sec at maximum speed to elute the cDNA.
- 10. Couple cyanine dyes to the aa-dUTP incorporated cDNAs by adding the eluted material directly to 3 L of Cy5 or Cy3 dye resuspended in DMSO. Incubate for 1 h to overnight at room temperature in a lightproof box or drawer.
- 11. Purify the cDNA using a MinElute kit, following the manufacturer's instructions.
The products of this protocol will be Cy5-labeled and Cy3-labeled cDNA, which are ready to hybridize to microarrays. This material can be stored in foil, to prevent bleaching, at 4C for up to 1 wk. A useful check for labeling success is the color of the labeled cDNA after unincorporated nucleotides have been removed: Good labeling will result in visible blue (Cy5) or red (Cy3) color in the cleaned up material.
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.
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