Increasing specificity of real time PCR to detect microRNA through primer design and annealing temperature increase.
OBJECTIVE
To investigate the non-specific and inaccurate amplification in cases of highly similar sequences among family members and the length heterogeneity of mature microRNA (miRNA), and find a condition that discriminates maximally among similar miRNA family members and detects the accurate expression level of miRNAs.


METHODS
Primers with their mismatches and/or 3' end at different positions were designed. Amplification efficiencies were compared using matched and various mismatched primers by RNA-tailing and primer-extension RT-PCR at different annealing temperatures. Expression levels of several miRNAs in mouse brain were compared using miRNA specific primers with different termini.


RESULTS
Raising annealing temperatures 12 degrees Celsius-14 degrees Celsius above the T(m) of the primers maximally increased amplification specificity without sacrificing sensitivity. Primers designed with their termini on or near variant positions could efficiently discriminate between miRNA isoforms. Using primers that terminated before the end of the mature miRNA did not miss the detection of shorter mature miRNA and provided accurate expression levels.


CONCLUSIONS
Careful primer design and higher annealing temperature can increase specificity and accuracy of real time PCR miRNA detection.
He
Xiang-jun
XJ
Institute of Clinical Molecular Biology & Central Laboratory, Peking University People's Hospital, Beijing 100044, China.
Zhang
Qi
Q
Liu
Yu-jing
YJ
Pan
Xiu-ying
XY
D000818:Animals; D001921:Brain / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D035683:MicroRNAs / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013696:Temperature
D017931:DNA Primers; D035683:MicroRNAs