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/ Home / Protocols / cDNA Synthesis and Labelling

cDNA synthesis and labelling for mouse (Synapse chips)

Toutes les références et les fournisseurs mentionnés dans ce protocole sont ceux que nous utilisons et que nous recommandons. Il n’y a aucune obligation à utiliser ces produits ou fournisseurs en particulier.

cDNA labelling:

  1. Mix:
    • 10 µg RNA total
    • 5 µg random hexamers
    • 2 µg d’oligo dT
    • Qsp 23 µl water for Cy3 and 22 µl for Cy5.
  2. Make one reaction per RNA sample. Place the tubes in the PCR machine and start the following program:
    70 °C 10 minutes (RNA denaturation)
    4 °C 5 minutes
    Pause at 4 °C add the RT + Cy mix to each tube (see step 3)
    23 °C 10 minutes (annealing)
    42 °C 2 hours (elongation)
    Pause at 4 °C  
    37 °C 15 minutes or 70°C 10 minutes (RNA hydrolysis)
    Pause at 4 °C  
  3. While the tubes are incubating to 70°C, prepare the RT+Cy mixture, (be careful: Cy3 and Cy5 should not be mixed):
      Cy3 Cy5
    5x Superscript III buffer (Invitrogen) 8 µl 8 µl
    0,1 M DTT (Invitrogen) 4 µl 4 µl
    dNTP’s* 2 µl 2 µl
    Cy3 dUTP (1 mM) 1 µl -
    Cy5 dUTP (1 mM) - 2 µl
    Superscript III (Invitrogene, 200 U/ml) 2 µl 2 µl
    TOTAL 18 µl 17 µl
    * dNTPs concentration: 2,5 mM dA, dC and dGTP, 1,25 mM dTTP. Mix 25 ml of dA, dC and dGTP (100 mM), 12,5 ml of dTTP (100 mM) and 912,5 ml H20 Rnase/Dnase free.
  4. Once samples have cooled to 4°C following the 70°C incubation, add the appropriate labelling mix to each tube, pipetting up and down to mix.

Denaturation of RNA:

After the RT reaction, it is necessary to remove all traces of RNA and RNA/DNA hybrids. 2 techniques are possible:

RNAse treatment of probe

  1. Add:
    • 1 µl of RNAse H (Invitrogen, 1-4 U/ml).
    • 0.5 µl of RNAse A (20-30 U/µl).
  2. Incubate at 37°C for at least 15 minutes.

Chemical hydrolysis

Note: use freshly prepared NaOH and HCl solutions

  1. Add: 15 µl of NaOH 0.1 N to degrade DNA.
  2. Incubate at 70°C for at least 10 minutes.
  3. Neutralize with 15 µl of HCl 0.1 M.

Samples purification (optional):

After the RNase treatment, it is necessary to remove enzymes, dNTPs and unincorporated fluorochromes. A precipitation using ethanol improves the purification.

Note: if the initial RNA's quantity is low, this step is to be banned.

  1. Preheat the water at 37 °C.
  2. If necessary, transfer the sample in an eppendorf sterile tube (1.5 ml) to be able to centrifuge the tube at 4 °C.
  3. - For a reaction of 40 µl add:
    • 4 µl of sodium acetate 3 M, pH 5.2 (1/10th of volume).
    • 100 µl of 96 % ethanol (2.5 volumes).
    • Note: if chemical hydrolysis is used, the volume is 70 µl per reaction.
  4. Vortex and precipitate at –80 °C for 30 minutes or at –20 °C during at least 12 hours (overnight or over weekend).
  5. Centrifuge 30 minutes at 13.000 rpm.
  6. Remove ethanol delicately and tap it slowly on a paper tissue. The pellet should not dry completely.

Probe purification (QuiaQuick columns from Quiagen) - for purified samples:

  1. Add 40 µl of water per tube.
  2. A dd 4 µl of sodium acetate 3 M, pH 5.2.
  3. A dd 200 µl of Qiagen buffer PB, then vortex briefly.
  4. If the pellet does not redissolve, place at 37°C for 15 minutes and vortex. If it is not sufficient, place at 95 °C for 30 seconds and vortex.
  5. Apply onto a “Qiaquick PCR clean up” column (it is possible to apply 2 reactions by column).
  6. Centrifuge at 13.500 rpm for 1 minutes.
  7. Remove the eluate.
  8. Apply 600 µl of Buffer EP, then centrifuge at 13.500 rcf for 1 minute.
  9. Remove the eluate.
  10. Centrifuge at 13.500 rcf for 2 minutes to dry the filter.
  11. Remove the eluate.
  12. Elute by applying 30 µl of water, pH 8.0, preheated at 37 °C, onto the centre of the column and allow it to enter by gravity flow for 1 minute.
  13. Centrifuge at 13.500 rcf for 1 minute. If the column remains colored, repeat the elution step.

We recommend using the probe quickly. However it can be stored for 24 hours at 4 °C, without significant decrease of the signal.

Probe purification (QuiaQuick columns from Quiagen) - if purification step omitted:

  1. Mix both probes.
  2. Add 700 µl of Qiagen buffer PB, then vortex briefly.
  3. Centrifuge at 13.500 rcf for 1 minute.
  4. Add 750 µl of buffer EP, then centrifuge at 13.500 rcf for1 minute.
  5. Remove the eluate.
  6. Centrifuge at 13.500 rcf for 2 minutes to dry the filter.
  7. Elute by applying 30 µl of water, pH 8.0, preheated in 37 °C onto the centre of the column and allow it to enter by gravity flow for 1 minute.
  8. Centrifuge at 13.500 rcf for 1 minute. If the column remains colored, repeat elution step.

We recommend using the probe quickly. However it can be stored for 24 hours at 4 °C, without significant decrease of the signal.

To concentrate cleaned cDNA's, air-dry the tube in a speedvac until almost all the liquid has been eliminated (only a micro-drop should remain).





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