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PCR was performed with a 2 l cDNA mixture in a total volume of 35 l using Taq polymerase and 0.2 nmol/l of each forward and reverse primer (intron spanning, 5CCCCCGATAGCTCCTACAAC3 and 3AAGGCCTTCCTCATGCACT5, TRPV1 mRNA accession number NM_018727) after initial 95 C for 3 min over 35 cycles of 95 C for 30 s, 64 C for 30 s and 72 C for 45 s, finalized by 72 C for 7 min and a temperature hold at 4 C in a Techne TC-512 gradient thermal cycler (Staffordshire, UK), yielding a 295 bp product
they stopped, in other words, because the drug was too effective
There is no clinically proven best time
These results have been most evident over several months
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