Kevin Kuhlmann1, Melanie Cieselski1, Julia Schumann2. 1. University Clinic and Outpatient Clinic for Anesthesiology and Operative Intensive Care, University Medicine Halle (Saale), 06112, Franzosenweg 1a, Halle (Saale), Germany. 2. University Clinic and Outpatient Clinic for Anesthesiology and Operative Intensive Care, University Medicine Halle (Saale), 06112, Franzosenweg 1a, Halle (Saale), Germany. julia.schumann@uk-halle.de.
Abstract
BACKGROUND: In the present study, two distinct PCR methods were used for the quantification of genetic material and their results were compared: real-time-PCR (qPCR; relative quantification) and droplet digital PCR (ddPCR; absolute quantification). The comparison of the qPCR and the ddPCR was based on a stimulation approach of microvascular endothelial cells in which the effect of a pro-inflammatory milieu on the expression of vasoactive receptors was investigated. RESULTS: There was consistency in directions of effects for the majority of genes tested. With regard to the indicated dimension of the effects, the overall picture was more differentiated. It was striking that deviations were more pronounced if the measured values were on the extreme edges of the dynamic range of the test procedures. CONCLUSIONS: To obtain valid and reliable results, dilution series are recommended, which should be carried out initially. In case of ddPCR the number of copies per µl should be adjusted to the low three-digit range. With regard to qPCR it is essential that the stability and reliability of the reference genes used is guaranteed. Here, ddPCR offers the advantage that housekeeping genes are not required. Furthermore, an absolute quantification of the sample can be easily performed by means of ddPCR. Before using ddPCR, however, care should be taken to optimize the experimental conditions. Strict indications for this methodology should also be made with regard to economic and timing factors.
BACKGROUND: In the present study, two distinct PCR methods were used for the quantification of genetic material and their results were compared: real-time-PCR (qPCR; relative quantification) and droplet digital PCR (ddPCR; absolute quantification). The comparison of the qPCR and the ddPCR was based on a stimulation approach of microvascular endothelial cells in which the effect of a pro-inflammatory milieu on the expression of vasoactive receptors was investigated. RESULTS: There was consistency in directions of effects for the majority of genes tested. With regard to the indicated dimension of the effects, the overall picture was more differentiated. It was striking that deviations were more pronounced if the measured values were on the extreme edges of the dynamic range of the test procedures. CONCLUSIONS: To obtain valid and reliable results, dilution series are recommended, which should be carried out initially. In case of ddPCR the number of copies per µl should be adjusted to the low three-digit range. With regard to qPCR it is essential that the stability and reliability of the reference genes used is guaranteed. Here, ddPCR offers the advantage that housekeeping genes are not required. Furthermore, an absolute quantification of the sample can be easily performed by means of ddPCR. Before using ddPCR, however, care should be taken to optimize the experimental conditions. Strict indications for this methodology should also be made with regard to economic and timing factors.
Authors: Leonardo B Pinheiro; Victoria A Coleman; Christopher M Hindson; Jan Herrmann; Benjamin J Hindson; Somanath Bhat; Kerry R Emslie Journal: Anal Chem Date: 2011-12-21 Impact factor: 6.986
Authors: Benjamin J Hindson; Kevin D Ness; Donald A Masquelier; Phillip Belgrader; Nicholas J Heredia; Anthony J Makarewicz; Isaac J Bright; Michael Y Lucero; Amy L Hiddessen; Tina C Legler; Tyler K Kitano; Michael R Hodel; Jonathan F Petersen; Paul W Wyatt; Erin R Steenblock; Pallavi H Shah; Luc J Bousse; Camille B Troup; Jeffrey C Mellen; Dean K Wittmann; Nicholas G Erndt; Thomas H Cauley; Ryan T Koehler; Austin P So; Simant Dube; Klint A Rose; Luz Montesclaros; Shenglong Wang; David P Stumbo; Shawn P Hodges; Steven Romine; Fred P Milanovich; Helen E White; John F Regan; George A Karlin-Neumann; Christopher M Hindson; Serge Saxonov; Bill W Colston Journal: Anal Chem Date: 2011-10-28 Impact factor: 6.986
Authors: Coren A Milbury; Qun Zhong; Jesse Lin; Miguel Williams; Jeff Olson; Darren R Link; Brian Hutchison Journal: Biomol Detect Quantif Date: 2014-08-20