Use of Eppendorf LoBind® Tubes to consistently prepare and store standard panels for real-time PCR absolute quantifications

Applications Maxime Lecerf*, Jérôme Le Goff Université Paris V, Equipe “Immunité et Biothérapie Muqueuses”, Unité INSERM Internationale U743 (Immunologie Humaine), Centre de Recherches Biomédicales des Cordeliers, 15 rue de l’Ecole de Médecine, 75270 Cedex 06, Paris, France *Corresponding author e-mail address: maxime.lecerf@crc.jussieu. fr Technical Report Real-time PCR is a powerful technology for quantification of target nucleic acids sequences as it combines specificity and sensitivity. The so-called absolute quantification enables to determine DNA concentrations of samples by running standards in parallel. However, this requires strict setup of the PCR reaction and rigorous standard panel preparation in order to obtain reliable results [1]. Although primers and probe design, as well as PCR condition setup are often the trickiest steps, standards preparation must not be underestimated. Standard panels serve as a basis for calibration curve by plotting Ct values of the standards against log10 of corresponding amounts of DNA. The slope of this curve reflects the amplification efficiency and can be determined as follows: Eq. 1: Efficiency = 10 -1/Slope -1 Optimized PCR, being an exponential reaction at least in the beginning, with a theoretical maximum efficiency of 1.00 (or 100 %) means that amplicon quantity doubles at each cycle. Moreover, calibration curve also allows user to determine template concentration of samples from Ct values which were determined experimentally. Introduction Application Note 226 | Page 2 In this context, standard panel preparation appears as a crucial step in the reliable assessment of amplification efficiency and correct sample quantification. However, this preparation is often challenging because a good panel should range over several orders of magnitude of dilution (log10 steps) [1]. Dealing with very low concentrations of nucleic acid such as 10 copies of genomic DNA per 10 μL involves that sample adsorption on vessels cannot be ignored. Indeed, the loss of DNA on vessel surfaces will be amplified at each dilution step and will be particularly significant for the most diluted standards leading to a DNA concentration actually lower than expected. Accordingly, the observed amplification efficiency will be significantly reduced not because of a problem with the enzymatic reaction but simply because the initial DNA quantity is overestimated, leading to misinterpretation of the results. The following experiment will describe how the choice of laboratory vessel influences the apparent real-time PCR efficiency by preparing serial dilution of standard panels and/or storing them on Eppendorf DNA LoBind or competitor low retention tube. Nucleic acid purification: Genomic DNA was obtained from lymphoblastoid T-cell line 8E5 (NIH; ATCC 8993). Cultured cells were harvested, counted under microscope and resuspended in PBS at 5 x 106 cells/mL. DNA was extracted from 200 μL of cell suspension using the QIAamp® DNA Blood Mini Kit (Qiagen, Germany) according to manufacturer’s protocol. Elution was made in 200 μL of water (Biotechnology Performance Certified, SigmaAldrich, USA) to obtain a stock solution at 1 x 105 copies of genomic DNA per 10 μL. Genomic DNA panel: Serial 10-fold dilutions were carried out as follows in 1.5 mL tubes (either Eppendorf DNA LoBind or competitor low retention tubes): 100 μL of genomic DNA solution was transferred in a tube containing 900 μL of water according to Eppendorf pipetting guidelines. Tube was then vortexed for one minute and served as start solution for the next dilution step. Then, each dilution was aliquotted by 22 μL either on Eppendorf DNA LoBind tubes or competitor low retention tubes and stored at -80 °C for 24 hours (Figure 1). Quantification of genomic DNA target: Target gene (ALB) was quantified by real-time PCR. The forward primer was 5’-GCTGTCATCTCTTGTGGGCTGT -3’, the reverse primer was 5’AAACTCATGGGAGCTGCTGGTT -3’ and the probe was 5’FAM CCTGTCATGCCCACACAAATCTCTCC TAMRA -3’ [2]. Primers and probes were synthesized by Eurogentec (Belgium). Real-time PCR was carried out on a LightCycler® instrument using the LightCycler® FastStart® DNA Master HybProbe® kit (Roche Applied Science, Switzerland) [3]. 20 μL capillaries contained 1 x FastStart® DNA Master HybProbe buffer, additional 3 mM MgCl2, 0.3 μM each primer and probe and 10 μL of genomic DNA standard. Thermocycling conditions consisted of an initial step (95 °C for 10 min) and 45 cycles of denaturation (95 °C for 10 sec) and an annealing and polymerization step (60 °C for 30 s). All three standard panels were assayed in the same experiment. Standard curve: Ct values were plotted versus the log10transformed expected concentration of target gene. Linear regression was applied to each scatterplot and slope was used to determine apparent amplification efficiency using equation