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paramagnetic oligo-dT beads. While the mRNA is bound to the beads, reaction buffers
can easily be changed without loss of mRNA or cDNA. This allows using optimal (i.e.,
high) concentrations of cDNA primers and nucleotides during cDNA synthesis without
interference with the subsequent tailing reaction. To avoid loss of transcripts, do not
contaminate the reaction with RNases because the mRNA holds the newly synthesized
cDNA to the bead. After cDNA synthesis and before starting the tailing reaction, the
unbound cDNA synthesis primers and unincorporated dNTPs have to be washed out.
Tailing is performed in a KH2PO4 buffer that, unlike the provided potassium-cacodylate
buffers, does not inhibit the subsequent PCR reaction, which is set up in the same reaction
tube without discarding the tailing buffer.
Random primers were originally used because they reduced the length of an amplicon
and allowed amplification of 5′-sequences. These random primers, combined with oligodT primers, slightly improve the results when single cells are used (CFL5 primer mix).
However, when higher cell numbers (>100) are used, it appears that random primers alone
work at least as well as the combination. For single cells, a random octamer increases the
average fragment length, compared to a random hexamer, by ∼100 to 200 bp. Due to the
increasing number of commercially available oligo arrays that are restricted to the 3′-end,
it might be advantageous to use oligo dT primers alone. The authors’ first experiments
indicate that the CFl5CT(24) primer should be used in this instance.
Materials
Oligo dT kit (Dynal) including:
Dynabeads Oligo (dT)25
Washing buffer containing LiDS
Lysis buffer
Phosphate-buffered saline (PBS; APPENDIX 2)
5× RT buffer (Life Technologies)
0.1 M DTT (Life Technologies)
10% (v/v) Igepal
cDNA synthesis primers:
For mRNA amplification for ≥100 cells:
CFL5C6: 5′-(CCC)5 GTC TAG ANN NNN N-3′ (200 µM)
For single cells and 5′ and 3′ coverage:
CFl5C8: 5′-(CCC)5 GTC TAG ANN NNN NNN-3′ (200 µM)
CFl5CT: 5′-(CCC)5 GTC TAG ATT TTT TTT TTT TTT TVN-3′ (100 µM)
CFL5 primer mix: 1 vol CFl5c8 (200 µM) + 1 vol CFl5cT (100 µM)
For the use of 3′-restricted oligo arrays:
CFl5CT(24): 5′-(CCC)5 GTC TAG ATT (T)22VN-3′
10 mM and 200 µM dNTPs
Reverse transcriptase (Superscript II; Life Technologies)
Igepal wash buffer (see recipe)
Tween 20 wash buffer (see recipe)
40 mM MgCl2
2 mM dGTP
200 mM KH2PO4
Tailing wash buffer (see recipe)
Mineral oil
Terminal deoxynucleotide transferase (TdT; Amersham Pharmacia Biotech)
Expand Long Template (ELT) PCR system (Roche Diagnostics) including:
10× ELT buffer 1 (17.5 mM MgCl2)
3.5 U/µl DNA polymerase mix
Discovery of
Differentially
Expressed Genes
25B.8.3
Current Protocols in Molecular Biology
Supplement 70