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cDNA synthesis and labelling for mouse (Synapse chips)

• cDNA labelling
• Denaturation of RNA
• Samples purification (optional)
• Probe purification (QuiaQuick columns from Quiagen) - for purified samples
• Probe purification (QuiaQuick columns from Quiagen) - if purification step omitted

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).