Efficient Normalization of qPCR Data

The THP-1 cell line was sub-cloned and one clone (#5) was selected for its ability to differentiate relatively homogeneously in response to phorbol 12-myristate-13-acetate (PMA) (Sigma). THP-1.5 was used for all subsequent experiments. THP-1.5 cells were cultured in RPMI, 10% FBS, Penicillin/Streptomycin, 10 mM HEPES, 1 mM Sodium Pyruvate, 50 uM 2-Mercaptoethanol. THP-1.5 were treated with 30 ng/ml PMA over a time-course of 96 h. Total cell lysates were harvested in TRIzol reagent at 1, 2, 4, 6, 12, 24, 48, 72, 96 hours, including an undifferentiated control. Undifferentiated cells were harvested in TRIzol reagent at the beginning of the LPS time-course. One biological replicate was prepared for each time point. Total RNA was purifed from TRIzol lysates according to manufacturer's instructions. Gene-specific primer pairs were designed using Primer3 software [21 (link)], with an optimal primer size of 20 bases, amplification size of 140 bp, and annealing temperature of 60°C. Primer sequences were designed for 2,396 candidate genes including four potential controls: GAPDH, beta actin (ACTB), beta-2-microglobulin (B2M), phosphoglycerate kinase 1 (PGK1). The RNA samples were reverse transcribed to produce cDNA and then subjected to quantitative PCR using SYBR Green (Molecular Probes) using the ABI Prism 7900 HT system (Applied Biosystems, Foster City, CA, USA) with a 384-well amplification plate; genes for each sample were assayed in triplicate. Reactions were carried out in 20 μL volumes in 384-well plates; each reaction contained: 0.5 U of HotStar Taq DNA polymerase (Qiagen) and the manufacturer's 1× amplification buffer adjusted to a final concentration of 1 mM MgCl₂, 160 μM dNTPs, 1/38000 SYBR Green I (Molecular Probes), 7% DMSO, 0.4% ROX Reference Dye (Invitrogen), 300 nM of each primer (forward and reverse), and 2 μL of 40-fold diluted first-strand cDNA synthesis reaction mixture (12.5 ng total RNA equivalent). Polymerase activation at 95°C for 15 min was followed by 40 cycles of 15 s at 94°C, 30 s at 60°C, and 30 s at 72°C. The dissociation curve analysis, which evaluates each PCR product to be amplified from single cDNA, was carried out in accordance with the manufacturer's protocol. Expression levels were reported as Ct values.
The large number of genes assayed and the replicates measures required that samples be distributed across multiple amplification plates, with an average of twelve plates per sample. Because it was envisioned that GAPDH would serve as a single-gene normalization control, this gene was included on each plate. All primer pairs were replicated in triplicates.
Raw qPCR expression measures were quantified using Applied Biosystems SDS software and reported as Ct values. The Ct value represents the number of cycles or rounds of amplification required for the fluorescence of a gene or primer pair to surpass an arbitrary threshold. The magnitude of the Ct value is inversely proportional to the expression level so that a gene expressed at a high level will have a low Ct value and vice versa.
Replicate Ct values were combined by averaging, with additional quality control constraints imposed by a standard filtering method developed by the RIKEN group for the preprocessing of their qPCR data. Briefly this method entails:
1. Sort the triplicate Ct values in ascending order: Ct₁, Ct₂, Ct₃. Calculate differences between consecutive Ct values: difference₁ = Ct₂ - Ct₁ and difference2 = Ct₃ - Ct₂.
2. Four regions are defined (where Region₄ overrides the other regions):
Region₁: difference ≤ 0.2
Region₂: 0.2 < difference ≤ 1.0
Region₃: 1.0 < difference
Region₄: one of the Ct values in the difference calculation is 40
If difference₁ and difference₂ fall in the same region, then the three replicate Ct values are averaged to give a final representative measure. If difference₁ and difference₂ are in different regions, then the two replicate Ct values that are in the small number region are averaged instead.
This particular filtering method is specific to the data set we used here and does not represent a part of the normalization procedure itself; Alternate methods of filtering can be applied if appropriate prior to normalization. Moreover while the presentation in this manuscript has used Ct values as an example, any measure of transcript abundance, including those corrected for primer efficiency can be used as input to our data-driven methods.