Detail
Article
Online article
FT
Medvik - BMC
  • Something wrong with this record ?

How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments

D. Svec, A. Tichopad, V. Novosadova, MW. Pfaffl, M. Kubista,

. 2015 ; 3 (-) : 9-16. [pub] 20150311

Language English Country Germany

Document type Journal Article

Persistent link   https://www.medvik.cz/link/bmc16019960

We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1-16 qPCR replicates per concentration and we tested 2-10 μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3-4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.

References provided by Crossref.org

000      
00000naa a2200000 a 4500
001      
bmc16019960
003      
CZ-PrNML
005      
20160913112050.0
007      
ta
008      
160722s2015 gw f 000 0|eng||
009      
AR
024    7_
$a 10.1016/j.bdq.2015.01.005 $2 doi
024    7_
$a 10.1016/j.bdq.2015.01.005 $2 doi
035    __
$a (PubMed)27077029
040    __
$a ABA008 $b cze $d ABA008 $e AACR2
041    0_
$a eng
044    __
$a gw
100    1_
$a Svec, David $u Institute of Biotechnology, Academy of Science of the Czech Republic, Prague, Czech Republic; TATAA Biocenter, Gothenburg, Sweden.
245    10
$a How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments / $c D. Svec, A. Tichopad, V. Novosadova, MW. Pfaffl, M. Kubista,
520    9_
$a We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1-16 qPCR replicates per concentration and we tested 2-10 μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3-4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.
655    _2
$a časopisecké články $7 D016428
700    1_
$a Tichopad, Ales $u Faculty of Medicine Pilsen, Charles University, Pilsen, Czech Republic.
700    1_
$a Novosadova, Vendula $u Institute of Biotechnology, Academy of Science of the Czech Republic, Prague, Czech Republic; TATAA Biocenter, Gothenburg, Sweden.
700    1_
$a Pfaffl, Michael W $u Physiology Weihenstephan, TUM - Technische Universität München, Freising, Germany.
700    1_
$a Kubista, Mikael $u Institute of Biotechnology, Academy of Science of the Czech Republic, Prague, Czech Republic; TATAA Biocenter, Gothenburg, Sweden.
773    0_
$w MED00189513 $t Biomolecular detection and quantification $x 2214-7535 $g Roč. 3, č. - (2015), s. 9-16
856    41
$u https://pubmed.ncbi.nlm.nih.gov/27077029 $y Pubmed
910    __
$a ABA008 $b sig $c sign $y a $z 0
990    __
$a 20160722 $b ABA008
991    __
$a 20160913112408 $b ABA008
999    __
$a ind $b bmc $g 1154630 $s 944488
BAS    __
$a 3
BAS    __
$a PreBMC
BMC    __
$a 2015 $b 3 $c - $d 9-16 $e 20150311 $i 2214-7535 $m Biomolecular detection and quantification $n Biomol Detect Quantif $x MED00189513
LZP    __
$a Pubmed-20160722

Find record

Citation metrics

Archiving options