NDLI: Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency

Content Provider	Springer Nature Link
Author	Ruijter, Jan M. Lorenz, Peter Tuomi, Jari M. Hecker, Michael Hoff, Maurice J. B.
Copyright Year	2014
Abstract	The analysis of quantitative PCR data usually does not take into account the fact that the increase in fluorescence depends on the monitoring chemistry, the input of ds-DNA or ss-cDNA, and the directionality of the targeting of probes or primers. The monitoring chemistries currently available can be categorized into six groups: (A) DNA-binding dyes; (B) hybridization probes; (C) hydrolysis probes; (D) LUX primers; (E) hairpin primers; and (F) the QZyme system. We have determined the kinetics of the increase in fluorescence for each of these groups with respect to the input of both ds-DNA and ss-cDNA. For the latter, we also evaluated mRNA and cDNA targeting probes or primers. This analysis revealed three situations. Hydrolysis probes and LUX primers, compared to DNA-binding dyes, do not require a correction of the observed quantification cycle. Hybridization probes and hairpin primers require a correction of −1 cycle (dubbed C-lag), while the QZyme system requires the C-lag correction and an efficiency-dependent C-shift correction. A PCR efficiency value can be derived from the relative increase in fluorescence in the exponential phase of the amplification curve for all monitoring chemistries. In case of hydrolysis probes, LUX primers and hairpin primers, however, this should be performed after cycle 12, and for the QZyme system after cycle 19, to keep the overestimation of the PCR efficiency below 0.5 %. Figure The qPCR monitoring chemistries form six groups with distinct fluorescence kinetics. The displacement of the amplification curve depends on the chemistry, DNA input and probe-targeting. The observed shift in Cq values can be corrected and PCR efficiencies can be derived.
Starting Page	1689
Ending Page	1696
Page Count	8
File Format	PDF
ISSN	00263672
Journal	Microchimica Acta
Volume Number	181
Issue Number	13-14
e-ISSN	14365073
Language	English
Publisher	Springer Vienna
Publisher Date	2014-01-14
Publisher Place	Vienna
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Quantitative PCR Monitoring chemistry DNA-binding dyes Hydrolysis probes Hybridization probes PCR efficiency Nanochemistry Nanotechnology Characterization and Evaluation of Materials Analytical Chemistry Microengineering
Content Type	Text
Resource Type	Article
Subject	Analytical Chemistry

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency

Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency

DNA biosensors based on metallo-intercalator probes and electrocatalytic amplification

Microfluidic high-throughput reverse-transcription quantitative PCR analysis of liver gene expression in lactating animals

Electrokinetically-controlled RNA-DNA hybridization assay for foodborne pathogens

Dendrimer-based biosensor for chemiluminescent detection of DNA hybridization

Luminescent probes for detection and imaging of hydrogen peroxide

CdTe-CdSe nanocrystals capped with dimethylaminoethanethiol as ultrasensitive fluorescent probes for chromium(VI)

Calcein-functionalized Fe3O4@SiO2 nanoparticles as a reusable fluorescent nanoprobe for copper(II) ion

Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency

Similar Documents

Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency

Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency

DNA biosensors based on metallo-intercalator probes and electrocatalytic amplification

Microfluidic high-throughput reverse-transcription quantitative PCR analysis of liver gene expression in lactating animals

Electrokinetically-controlled RNA-DNA hybridization assay for foodborne pathogens

Dendrimer-based biosensor for chemiluminescent detection of DNA hybridization

Luminescent probes for detection and imaging of hydrogen peroxide

CdTe-CdSe nanocrystals capped with dimethylaminoethanethiol as ultrasensitive fluorescent probes for chromium(VI)

Calcein-functionalized Fe3O4@SiO2 nanoparticles as a reusable fluorescent nanoprobe for copper(II) ion

Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency