mir-16-5p as a Suitable Reference Gene for Normalization of Quantitative Real Time PCR in Acute Lymphoblastic Leukemia

• Published in: Pakistan journal of zoology Vol. 51; no. 2; p. 747
• Main Authors: Shahid, Samiah, Shaheen, Jawaria, Shahid, Wajeehah, Akhtar, M. Waheed, Sadaf, Saima
• Format: Journal Article
• Language: English
• Published: Lahore Knowledge Bylanes 30.04.2019; The Zoological Society of Pakistan
• Subjects: Acute lymphoblastic leukemia; Acute lymphocytic leukemia; Algorithms; Anopheles; Biochemistry; Cancer; Cancer genetics; Cancer research; Chromosome 5; Diagnosis; Experiments; Gene expression; Genes; Leukemia; Lymphatic leukemia; Lymphocytic leukemia; Medical examination; Messenger RNA; Methods; MicroRNA; MicroRNAs; miRNA; Polymerase chain reaction; Real time; RNA; Stem cells; Studies; Pakistan; Lahore Pakistan; Germany
• ISSN: 0030-9923
• DOI: 10.17582/journal.pjz/2019.51.2.747.754

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.