John M. S. Bartlett.pdf - Bio-Nica.info

cDNA Libraries from Few Cells 501
Fig. 2. Oligonucleotides used for 3′ anchored PCR. The three oligonucleotides A3′_1,2,3
are designed from A3′ NV and RA3′ NV to be used in PCR experiments. Note that these three
primers have to be used with A5′_1,2,3.
1.3. ss-cDNA Synthesis
The constraint caused by the size limitation of the ss-cDNA forbids the use of
oligo-dT to prime the reverse transcription. If such a priming strategy is chosen, this
will lead to a 3′-UTR-cDNA library. To obtain a cDNA library representative of the
sequences of all messenger RNA, priming with random primer RA3′ NV must be
performed.
Experimentally, the ss-cDNA is synthesized with a random primer RA3′ NV (Fig. 2)
and a radiolabled nucleotide. The average size is determined by alkaline gel electrophoresis
and autoradiography. The incubation time with the reverse transcriptase
is calibrated in order to generate an ss-cDNA whose average length is between 0.8
and 1 kbp. With those conditions the representation of any mRNA will be optimal
in the library.
1.4. Amplification of the cDNA Library Based on the SLIC Strategy
We have used this strategy to generate a cDNA library from newborn rat cervical
superior ganglia (CSG). Total RNA was prepared from one single CSG, and polyA +
RNA was prepared with oligo dT-coated magnetic beads (Dynabeads mRNA purification
kit). Half of the material, which corresponds to polyA + RNA of about 5000 cells,
was used to synthesize ss-cDNA primed with RA3′ NV. An incubation time of 30 min
was optimum to generate an average size of 1 kbp. After removal of the primer, the
ss-cDNA was ligated to A5′ NV (note that after each step the different primers are
removed). These ss-cDNA were amplified by two rounds of nested PCR. To increase
the specificity of the PCR reaction, we have used the touchdown PCR protocol (3). The
primers used for the nested PCR were A5′_1 ∞ A3′_1 and A5′_2 ∞ A3′_2, respectively.
One-twentieth of the reaction was cloned in a blunt-end vector, yielding 2 ∞ 10 5
colonies. Analysis by direct PCR on colonies of 96 randomly chosen clones indicated
that the average size of the library was about 900 bases. The striking result is that the