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230 Dominguez et al.
Fig. 2. cDNA normalization for their concentration in GAPDH gene. Lanes 1, 3, 5, and
7 cDNA diluted 15 are different samples and. Lanes 2, 4, 6, and 8 are the respective cDNA
diluted 150. Lane 9 is a PCR-negative control.
3.2. cDNA Synthesis and Further Treatment
1. A single reverse transcription with any of the two RT primers shown in Table 1 (G7AU2dT
and GTGAU2dT) produced cDNA for a complete set of AU-DD reactions with matching
PCR primers. These two different RT primers are anchored oligo(dT) 15 primers with a 5′ tag
that accommodates the sequence of any of the AU-DD primers that will be used at the PCR
step. The tag defines the particular RT primer and the set of AU-DD primers to be used. The
15 T stretch was found to anneal more specifically on poly-A tails at the conditions used
than others of 25 or longer. The tag was designed with the intention of generating the
weaker secondary structure as possible. Standard oligo(dT) 15 was used to retrotranscribe
control RNA samples (Jurkat and U937). First-stranded cDNA was prepared with
Superscript II (Gibco-BRL) following manufacturer instructions with minor modifications
indicated below.
2. DNase treated total RNA (2–3 µg) in water and 10 pmoles of the chosen RT primer (Table 1)
for a reaction volume of 20 µL were denatured at 72°C for 3 min on a PCR machine and
chilled on ice for 1 min (see Note 10).
3. The other reagents for a reaction volume of 20 µL except the enzyme are supplemented
at room temperature (see Note 11). Annealing was allowed to proceed for 10 min at
room temperature.
4. 200 U SuperScript II (Gibco-BRL) were then added and the mixture was incubated for
1 h at 42°C.
5. Reverse transcription was stopped by heating at 90°C for 2 min and RNAse H was used as
recommended by its supplier (Gibco-BRL). 1.8 U RNase H, 20 min at 37°C.
6. To validate cDNA synthesis, normalization was performed by amplifying 110 and
1500 cDNA dilutions for GAPDH in a 25-cycle PCR adjusted to an annealing temperature
of 60°C (primers in Table 1B). Product concentration was estimated by visual inspection
of ethidium bromide stained gels, and cDNAs were normalized by dilution according
to their GAPDH equivalents if required. Typically no adjustment was needed (Fig. 2,
see Note 12).
7. Free nucleotides and primer were washed out in Qiaquick columns (Qiagen). cDNA was
eluted in 50 µL of EE. The eluates were used directly in AU-DD.