SuperScript Indirect cDNA Labeling from total RNA

Modified by Rob Alba on 07/07/05

First-Strand cDNA Synthesis using Invitrogen's SuperScript Indirect cDNA Labeling Kit (Catalog numbers L1014-01 and L1014-02).

What follows is the protocol I use for cDNA synthesis from total RNA and indirect cDNA labeling prior to microarray hybridizations. Although I have made a few non-trivial modifications, the protocol below is similar to that in the instruction manual provided with this kit.

First-Strand cDNA Synthesis Rxn.

  1. Mix and briefly spin each kit component before use.
  2. Prepare rxns as follows:
    X µl DEPC-H2O
    X µl total RNA (15 to 20 µg/rxn; typically I use 15 µg/rxn)
    2 µl Anchored Oligo(dT)20 Primer (2.5µg/ µl)
                       Total Volume = 18 µl
  3. Incubate tubes at 70oC for 10 min, and then place on ice for 5 min.
  4. Add the following to each rxn tube on ice:
    6 µl 5X First-Strand buffer
    1.5 µl 0.1 M DTT
    1.5 µl 10-mM dNTP mix
    1 µl RNaseOUT (40 µl)
    2 µl SuperScript III RT (400 µl)
                       Total Volume = 30 µl
  5. Mix gently and spin briefly. Incubate tube at 46oC.
  6. Add 15 µl of 1 N NaOH to each rxn tube and mix thoroughly.
  7. Incubate tube at 70oC for 10 min.
  8. Add 15 µl of 1N HCl; mix gently.
  9. Add 20 µl 3M sodium acetate (pH 5.2); mix gently.
Purifying First-Strand cDNA.
  1. Add 500 µl of Loading Buffer to the cDNA (from Step 9) and mix well.
  2. Place a SNAP Column on a collection tube and load your cDNA on the column.
  3. Spin at 14,000g at room temp for 1 min; discard the flow-through.
  4. Place the SNAP Column onto the same collection tube and add 500 µl of Wash Buffer.
  5. Spin at 14,000g at room temp for 1 min; discard the flow-through.
  6. Repeat Steps 4 and 5 twice more, for a total of three 500 µl washes.
  7. Spin one more time at 14,000g at room temp for 1 min sec; discard the flow-through.
  8. Place the SNAP Column onto a new 1.5-ml tube.
  9. Add 50 µl of DEPC-treated water to the SNAP Column and incubate at room temp for 1 min. Elute the cDNA via spin at 14,000g at room temp for 1 min.
  10. Repeat Step 9, using the same 1.5-ml tube.
  11. Add 10 µl of 3 M sodium acetate (pH 5.2) to the eluent from steps 9 and 10.
  12. Add 2 µl of glycogen (20mg/ml) to the tube and mix.
  13. Add 300 µl of ice-cold 100% EtOH, and incubate the tube for 45 min to 1hr at -80oC.
  14. Spin the tube at 14,000 g at 4oC for 20 min. Carefully remove the supernatant.
  15. Add 500 µl of ice-cold 70% EtOH and spin the tube at 14,000g at 4oC for 5 min. Carefully remove the supernatant.
  16. Air dry the sample for ~5 min; ensure that all EtOH is removed.
  17. Warm the 2X Coupling Buffer at 37oC for 5 min and re-suspend the cDNA sample in 5 µl of warm 2X Coupling Buffer. Heat the cDNA/Coupling buffer at 50oC for 10 min and vortex well. Ensure that your cDNA pellet is fully re-suspended in the 2X Coupling Buffer.
Labeling with Fluorescent Dye. When preparing the rxn, be careful to minimize exposure of the dye solution to light. Also, DMSO is hygroscopic and will absorb moisture from the air, which will react with the NHS ester of the dye and significantly reduce the coupling rxn efficiency. Keep the DMSO supplied in the kit in an amber screw-capped vial at -20oC, and let the vial warm to room temperature before opening to prevent condensation. Use only the DMSO provided with this kit.
  1. Open one packet of Cy3- or Cy5- dye and add 75 µl of DMSO directly to the dye vial.
  2. Add 5 µl of the DMSO/dye solution to the tube from Step 17.
  3. Mix well and incubate the tube at room temp in the dark for 1 hr.
  4. Add 20 µl of 3 M Sodium Acetate (pH 5.2) to the dye-coupled cDNA solution.
  5. Add 500 µl of Loading Buffer to the cDNA solution. Mix well by vortexing.
  6. Place a SNAP Column onto a clear collection tube and load the cDNA/buffer solution.
  7. Spin at 14,000g at room temp for 1 min; discard the flow-through.
  8. Place the SNAP Column on the same collection tube; add 500 µl of Wash Buffer to column.
  9. Spin at 14,000g at room temp for 1 min; discard the flow-through.
  10. Repeat Steps 8-9 three times, for a total of four 500 µl washes.
  11. Spin one more time at 14,000g at room temp for 1 min; discard the flow-through.
  12. Place the SNAP Column onto a new amber collection tube.
  13. Add 63 µl of DEPC-water to the SNAP Column and incubate at room temp for 1 min. Spin at 14,000g at room temp for 1 min and collect the flow-through. The flow-through should contain 60 µl of your purified dye-coupled cDNA.
Assessing the Labeling Procedure. Conduct a spec assay to assess each Cy-cDNA labeling reaction using the procedure described in TIGR's Standard Operating Proceedure #M004 (http://pga.tigr.org/sop/M004_1a.pdf). This technique is also described briefly in the Appendix of the Instruction Manual for the Invitrogen cDNA labeling kit. Calculate the total pmol of synthesized cDNA, the total pmol of incorporated Cy dye for each labeling reaction, and the nucleotide/dye ratio for each reaction. Optimal labeling reactions generate >6000 pmols of cDNA (per 60 µl), >200 pmol of Cy dye (per 60 µl), and a nucleotide/dye ratio that is <30.