Design primers

As a result of this preliminary work, the sequence available for designing primer to quantify the expression of P-actin is restricted to two regions, between nucleotides 1 and 90 or between nucleotides 1208 and 1763. There is no exon boundary in the second region. The design should, therefore, be limited to the region between the first and second exons of the gene. Transfer this sequence to the primer design software (e.g. Primer Express® from ABI) and proceed to design.

Copy the mRNA sequence into a simple PCR document. Restrict the target sequence accordingly. Select the option for primer Tm (59°C) and amplicon length (80 to 120). Keep the CG content at default settings and primer length below 24 bp.

See if the software comes up with a possible primer pair. If not, use the primer test document and try to select sections of sequence manually. This sequence is very CG rich, therefore, select a region with A or T as 3' for each primer. Elongate the primer in 5' until reaching a Tm between 58° and 60°C.

Lastly, BLAST the primers (limit to the human species and mRNA bank). If the restriction in the target position has been done thoroughly, the BLAST search should give no matches. Quite a lot of hits are nevertheless reported for these primers. Most of them are on anti-sense strands of cDNAs. If none of them comes in both the forward and the reverse primer BLAST searches, no PCR product can be generated from these hits.

These primers are located in the 5' of the mRNA. This is an issue if the quality of the RNA is not sufficient to ensure reverse transcription up to the 5' end of the mRNA when an oligo-dT cDNA synthesis method is used. Therefore, a random hexamer cDNA synthesis is recommended when using these primers for P-actin as a housekeeping gene.

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