title: Fluorescence acquisition during hybridization phase in quantitative real-time PCR improves specificity and signal-to-noise ratio.
abstract: Quantitative real-time PCR (qPCR) is a standard method used for quantification of specific gene expression. This utilizes either dsDNA binding dyes or probe based chemistry. While dsDNA binding dyes have the advantage of low cost and flexibility, fluorescence due to primer dimers also interferes with the fluorescence of the specific product. Sometimes it is difficult, if not impossible, to standardize conditions and redesign primers in such a way that only specific fluorescence of the products of test and reference genes are acquired. Normally, the fluorescence acquisition in qPCR using dsDNA binding dyes is done during the melting phase of the PCR at a temperature between the melting points of primer dimers and the specific product. We have modified the protocol to acquire fluorescence during the hybridization phase. This significantly increased the signal-to-noise ratio and enabled the use of dsDNA binding dyes for mRNA quantification in situations where it was not possible when measurement was done in the melting phase. We have demonstrated it for three mRNAs, E6, E7, and DNMT1 with beta-actin as the reference gene, and for two miRNAs. This modification broadens the scope of qPCR using dsDNA binding dyes.
authors.affiliation: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Mohit
authors.initials: M
authors.lastname: Mehndiratta
authors.forename: Jayanth Kumar
authors.initials: JK
authors.lastname: Palanichamy
authors.forename: Pradeep
authors.initials: P
authors.lastname: Ramalingam
authors.forename: Arnab
authors.initials: A
authors.lastname: Pal
authors.forename: Prerna
authors.initials: P
authors.lastname: Das
authors.forename: Subrata
authors.initials: S
authors.lastname: Sinha
authors.forename: Parthaprasad
authors.initials: P
authors.lastname: Chattopadhyay
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D015342:DNA Probes; D003627:Data Interpretation, Statistical; D005453:Fluorescence; D015870:Gene Expression; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D000199:Actins; D015342:DNA Probes; D035683:MicroRNAs; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human