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/ Home / Protocols / cDNA Synthesis and Labelling

cDNA synthesis and labelling for mouse (22k chips)

cDNA labelling:

Solutions:

  • Random Hexanucleotides (RPO) 50 µM.
  • Rtases Reverse-iT (ABgene), 5x buffer and DTT 0,1 M.
  • dNTP set 100 mM (Amersham):
    • Prepare dNTP mix:
      • dATP , dCTP, dGTP 25 µl (2,5 mM)
      • dTTP 12,5 µl (1,25 mM)
      • H20 912,5 µl
  • dUTP fluorescent : Amersham 1 mM (Cy3 dUTP PA53022, Cy5 dUTP PA55022).

Denaturation of RNA and hybridization of RPO:

    • mRNA 0,5-2 µg / total RNA 10-20 µg
    • RPO 6 µl
    • H2O to 23 µl
  • Denature 10 minutes at 70 °C in a PCR machine. Cool to 4 °C.

Preparation of mix - per tube:

  • 8 µl RT buffer 5x
  • 2µl DTT 0,1 M
  • 2 µl H2O
  • 2 µl dNTP mix
  • 2 µl Reverse Transcriptase

Labelling of cDNA:

  • After denaturation of ARNm, centrifuge.
  • Add 1 µl of fluorescent dUTP (Cy3 for the controle and Cy5 for the sample).
  • Add 16 µl of mix, agitate, centrifuge.
  • Incubate 10 minutes at 23 °C, then 2 hours at 42 °C (or overnight at 37 °C).
    (Optional: add 1 µl of enzyme per tube and put back for 1 hour at 42 °C).

Denaturation of mRNA:

  1. Add 15 µl of NaOH 0.1 N and heat 10 minutes at 70 °C.
  2. Neutralize with 15 µl HCl 0.1 N.

Purification on Qiaquick PCR purification kit (Qiagen, ref.28106):

  1. Mix both targets Cy3 et Cy5.
  2. Add 5 volumes of buffer PB per tube, vortex and centrifuge briefly.
  3. Poor in a Qiaquick column, centrifuge 13000 rpm 1 minute.
  4. Wash the column with 750 µl buffer PE made with ethanol 95°, centrifuge.
  5. Recentrifuge.
  6. Elute with 30 µl of Tris 10 mM filtered. Wait 1 minute then centrifuge 13000 rpm 1 minute.




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