mir-16-5p as a Suitable Reference Gene for Normalization of Quantitative Real Time PCR in Acute Lymphoblastic Leukemia
mir-16-5p as a Suitable Reference Gene for Normalization of Quantitative Real Time PCR in Acute Lymphoblastic Leukemia
Samiah Shahid1,Jawaria Shaheen1, Wajeehah Shahid2, M. Waheed Akhtar1,3 and Saima Sadaf1,*
ABSTRACT
Quantitative real time polymerase chain reaction PCR (qPCR) may be utilized as a sensitive and reliable technique for the determination of circulating miRNA expression. Despite recent advancements, there is not a single consensus on the use of reference gene for quantification of circulating miRNAs through qPCR analyses in ALL. In the current study, we identified the reference gene that is the most suitable for qPCR normalization in patient and control plasma samples of ALL. Three highly reported reference genes namely RNU6-2, mir-16-5p and cel-mir-39-1 were selected as the candidate genes for normalization. Preliminary, quantification was performed through real time PCR in eight samples. The geNorm algorithm and comparative delta Cq method were used for selection of suitable reference gene out of the three candidate genes. The validation studies were, thereafter, performed on 112 samples including 87 patients and 25 normal healthy controls. The geNorm algorithm exhibited circulating mir-16-5p with the highest expression stability demonstrated by geNorm M value of 0.418. The comparative delta Cq method showed no significant difference in Cq values of ALL patients and healthy controls suggesting that mir-16-5p is the most stable normalizer for miRNA expression profiling assays in ALL. The results revealed that circulating mir-16-5p may be utilized as a suitable reference gene for normalization and improved quantification of miRNA.
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April 2019
Vol. 51, Iss. 2, Pages 399-799
