The British journal of dermatology · 2013
MicroRNA normalization candidates for quantitative reverse-transcriptase polymerase chain reaction in real time in lesional and nonlesional psoriatic skin.
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Langkilde A, Raaby L, Johansen C, Iversen L
Аннотация
BACKGROUND: MicroRNA (miRNA) technology is an evolving research area and quantitative reverse-transcriptase polymerase chain reaction in real time (qPCR) is an important investigational tool. The small noncoding RNA candidates used for normalization in psoriatic skin biopsies have never been sufficiently validated.
OBJECTIVES: To identify a reliable normalization method for miRNA analysis with qPCR in psoriatic skin.
METHODS: Five miRNA candidates previously used for normalization in psoriatic skin were identified through a search of the literature. Five other candidates were uncovered using the NormFinder algorithm on miRNA microarray data. The candidates were validated with qPCR in paired psoriatic biopsies, biopsies from patients during treatment and normal healthy skin. The stability of the miRNAs was determined with NormFinder and geNorm. The dispersion of data was determined before and after use of different normalization approaches.
RESULTS: In lesional and nonlesional skin the two algorithms ranked the candidates similarly, with an excellent correlation (R(2) = 0·95). miR-26a had the best stability, whereas the commonly used RNU48 had less favourable stability. The same results were seen within the dispersion of the data, including in biopsies from lesional, nonlesional, treated psoriatic and normal healthy skin.
CONCLUSIONS: This is the first study to validate the reliability of miRNA candidates for normalization by qPCR in psoriatic skin. From this study we conclude that miR-26a is the best candidate, and better than those previously used. miR-26a can be used for miRNA normalization in future studies with psoriatic skin.
OBJECTIVES: To identify a reliable normalization method for miRNA analysis with qPCR in psoriatic skin.
METHODS: Five miRNA candidates previously used for normalization in psoriatic skin were identified through a search of the literature. Five other candidates were uncovered using the NormFinder algorithm on miRNA microarray data. The candidates were validated with qPCR in paired psoriatic biopsies, biopsies from patients during treatment and normal healthy skin. The stability of the miRNAs was determined with NormFinder and geNorm. The dispersion of data was determined before and after use of different normalization approaches.
RESULTS: In lesional and nonlesional skin the two algorithms ranked the candidates similarly, with an excellent correlation (R(2) = 0·95). miR-26a had the best stability, whereas the commonly used RNU48 had less favourable stability. The same results were seen within the dispersion of the data, including in biopsies from lesional, nonlesional, treated psoriatic and normal healthy skin.
CONCLUSIONS: This is the first study to validate the reliability of miRNA candidates for normalization by qPCR in psoriatic skin. From this study we conclude that miR-26a is the best candidate, and better than those previously used. miR-26a can be used for miRNA normalization in future studies with psoriatic skin.