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To achieve maximal sensitivity, conditions were sought to avoid unnecessary loss of
mRNA during the precipitation steps. Enzymatic activity of the reverse transcriptase or
Taq polymerase should not be compromised by using less than an optimal supply of
substrates or by inadequate buffers.
The basic goal of this protocol is to introduce two binding sites for PCR primers into
cDNAs representing transcripts, allowing amplification of each transcript uniformly (Fig.
25B.8.1). The first primer-binding site is contained within a flanking region that lies at
the 5′-end of a random cDNA synthesis primer or an oligo dT primer. The second is
introduced through a tailing step using terminal deoxynucleotide transferase (TdT).
Therefore, three enzymatic steps are required—cDNA synthesis, tailing, and PCR. The
use of a random primer has two advantages. First, it enables amplification of 5′ regions
that might be of interest (e.g., when mutations are studied), and second, it leads to
production of cDNAs of lengths that are optimal for PCR amplification. However, for
cDNA synthesis with a random primer, it is important to remove most of the rRNA and
tRNA, which comprise >95% of total cellular RNA. Therefore, mRNA is purified using
1. mRNA isolation
(AAAAAAAAAAAAAAAAAAAAAAAAAAAAA)n
TTTTTTTTTTTTTTTTTTTTTTTTT
5′
2. primer-hybridization
(AAAAAAAAAAAAAAAAAAAAAAAAAAAAA)n
(TTTTTTT) 2TVN
TTTTTTTTTTTTTTTTTTTTTTTTT
5′- (CCC) 5
CFl5cT
NNNNNNNN
5′- (CCC) 5
5′
CFl5c8
3. cDNA-synthesis
(AAAAAAAAAAAAAAAAAAAAAAAAAAAAA)n
(TTTTTTT) 2TVN
TTTTTTTTTTTTTTTTTTTTTTTTT
5′
5′- (CCC) 5
4. RNA removal + G-tailing
(TTTTTTT)2TVN
GGGGGGGG(G)n
5′- (CCC) 5
5. CP2-PCR
5′ CCCCCCCCCCCCCCC
5′
CP2
(TTTTTTT) 2TVN
5′
CCCCCCCCCCCCCCC
GGGGGGGG(G)n
CP2
5′
CCCCCCCCCCCCCCC
CCCCCCCCCCCCCCC
Figure 25B.8.1 Global amplification of mRNA from a few or single cells. mRNA is captured by paramagnetic
beads (1), and primed using random and oligo dT primers containing a poly C flanking region (2). cDNA synthesis
starts from both primers (3; CFL5c8 is omitted in 3 and 4). After RNA removal, a poly G tail is added by TdT.
Using the poly C containing CP2 primer, all sequences can be amplified (5).
25B.8.2
Supplement 61
Current Protocols in Molecular Biology