Valid gene expression normalization by RT-qPCR in studies on hPDL fibroblasts with focus on orthodontic tooth movement and periodontitis

Meaningful, reliable and valid mRNA expression analyses by real-time quantitative PCR (RT-qPCR) can only be achieved, if suitable reference genes are chosen for normalization and if appropriate RT-qPCR quality standards are met. Human periodontal ligament (hPDL) fibroblasts play a major mediating role in orthodontic tooth movement and periodontitis. Despite corresponding in-vitro gene expression studies being a focus of interest for many years, no information is available for hPDL fibroblasts on suitable reference genes, which are generally used in RT-qPCR experiments to normalize variability between samples. The aim of this study was to identify and validate suitable reference genes for normalization in untreated hPDL fibroblasts as well as experiments on orthodontic tooth movement or periodontitis (Aggregatibacter actinomycetemcomitans). We investigated the suitability of 13 candidate reference genes using four different algorithms (geNorm, NormFinder, comparative ΔCq and BestKeeper) and ranked them according to their expression stability. Overall PPIB (peptidylprolyl isomerase A), TBP (TATA-box-binding protein) and RPL22 (ribosomal protein 22) were found to be most stably expressed with two genes in conjunction sufficient for reliable normalization. This study provides an accurate tool for quantitative gene expression analysis in hPDL fibroblasts according to the MIQE guidelines and shows that reference gene reliability is treatment-specific.


Supplementary Table 1. MIQE checklist for authors, reviewers and editors. E = essential information; D = desirable information.
Item to check Importance Description how item was addressed in study / article

Experimental design
Definition of experimental and control groups E Control group: untreated hPDL fibroblasts (physiological conditions); Experimental groups: hPDL fibroblasts treated with compressive orthodontic force (model for orthodontic tooth movement) or bacterial lysate of Aggregatibacter actinomycetemcomitans (Agac, model for bacterial periodontitis) for 24h. For details see materials and methods and Figure 5.
Number within each group E n = 6 Assay carried out by the core or investigator´s laboratory? D All assays were carried out in investigators' laboratory.
Acknowledgment of authors´ contributions D C.K. conceived the idea of the study/study design as well as designed/validated the used primer pairs. S.B., P.P. and A.S. contributed to discussion and study design. A.S. and C.K. conducted the experiments. A.S., C.K. and S.B. analysed the results. J.K. produced and contributed the Agac bacterial lysate. G.S. provided the primary hPDL fibroblasts. C.K. and A.S. wrote the manuscript and created the figures, tables and the supplementary material. All authors reviewed the manuscript.

Sample
Description E Primary human periodontal ligament fibroblasts (hPDL) were cultivated from periodontal connective tissue isolated from the middle root section of human teeth free of decay, which had been freshly extracted for medical reasons. A pool of hPDL cell lines from four different patients was used (1 male, 3 female, age: 16-23 years). Cells were identified by means of hPDL-specific marker gene expression and their spindle-shaped morphology (Supplementary Table 5 and Supplementary Figure). Ethical consent was obtained from the local ethics committee (12-170-0150).
Volume/mass of sample processed D Varying size of tissue sample / number of hPDL fibroblasts extracted. 70.000 cells were finally seeded per well / biological replicate for the experiments.

Microdissection or macrodissection E Microdissection
Processing procedure E Tissue samples were grown in 6-well cell culture plates until proliferation of adherently growing hPDL under normal cell culture conditions (37°C, 5% CO2, water-saturated) in full media, then trypsinized and further cultivated and passaged until the 6 th passage.
If frozen, how and how quickly? E Until use hPDL fibroblasts were frozen in liquid nitrogen (90% FCS, 10% DMSO, freezing 1°C/minute in cryo-box with isopropanol).
If fixed, with what and how quickly? E Not fixed.
Inhibition testing (Cq dilutions, spike, or other) E For evaluation of qPCR and primer efficiency as well as absence of inhibitors a log10 serial dilution series of a random cDNA sample from the untreated group was amplified in triplet for each candidate reference gene and the limit of detection (LOD) as the highest dilution, at which 95% (all three) of the technical replicates are detectable (Cq values), was determined. A standard curve was created by linear regression of the resulting Cq values with the relative dilution within the linear dynamic range (LDR) and the coefficient of determination r 2 as well as qPCR reaction efficiencies (E) with 95% confidence intervals were determined from the slope of the standard curve: E = (10 -1/slope -1) x 100%. Only primer pairs with a linear relation between Cq and log-transformed cDNA copy number (r 2 >0.98) were considered as possible valid reference gene candidates. In addition, only efficiencies E within the range of 90-110% were deemed acceptable. (Table 2, Supplementary Data 4)

Reverse transcription
Complete reaction conditions E To synthesize cDNA, we transcribed a standardized quantity of 1µg RNA per sample using a random hexamer primer (0.1 nmol, 1 µl, SO142, Life Technologies), an oligo-dT18 primer (0.1 nmol, 1 µl, SO131, Life Technologies, Thermo Fisher Scientific Inc.), 5× M-MLV-buffer (4 µl, M1705, Promega, Fitchburg, WI, USA) and dNTP mix (40 nmol, 1 µl, 10 nmol/dNTP, Roti ® -Mix PCR3, L785.2) ad 20 µl nuclease-free H2O (Roth BioScience Grade T143, Carl Roth GmbH & Co. KG). After incubation for 3 min at 70°C the mixture was quickly cooled on ice (RNA denaturation). We then added reverse transcriptase (200 U, 1 µl, M1705, Promega) and an RNase inhibitor (40 U, 1 µl, EO0381, Life Technologies), continued incubation at 37°C for 60 min and inactivated the reverse transcriptase by heat (95°C, 2 min). To minimize experimental variations, synthesis of cDNA, which was stored at −20°C until use, was performed concurrently for all samples. dCTP, dGTP, dTTP), stabilizers, Taq-DNA-polymerase (0.05 U/µl), JumpStart Taq antibody and SYBR Green I, as well as the respective cDNA-solution (1.5 µl, dilution 1:10) and the respective primer pair (7.5 pmol, 0.75 µl -3.75 pmol/primer) were pipetted ad 15 µl nuclease-free H2O (BioScience Grade T143, Carl Roth GmbH & Co. KG). A master-mix of all components except the cDNA solution was created to minimize technical errors during manual pipetting. We then amplified the cDNA in triplets (technical replicates) per candidate reference gene in 45 cycles (initial heat activation 95°C/5 min, per cycle 95°C/10 s denaturation, 60°C/8 s annealing, 72°C/8 s extension). At the end of each extension step SYBR Green I fluorescence was measured at 521 nm. For each biological replicate all genes were amplified in triplet on the same qPCR plate to minimize biasing effects of possible inter-run variations on relative reference gene stability assessment. Description of normalization method E Samples were not normalized, since apart from the reference genes no target genes were quantified.
Number and concordance of biological replicates D N = 1 (pool of hPDL fibroblasts from 4 different patients); n = 6 (pool cells seeded in 6 different wells per experimental group as biological replicates).
Number and stage (RT or qPCR) of technical replicates E qPCR reactions were performed in triplets (technical replicates n = 3).

Repeatability (intraassay variation) E
The maximum SD (of the mean) across all biological replicates (n=18) of the means of Cq from the three technical replicates was ≤0.553 in all instances.  Table 2) Reproducibility (interassay variation, CV) D High biological reproducibility was achieved as evidenced by the low SD of raw Cq values for all genes and experimental groups tested (see Figure 2, Supplementary Table 3).
Power analysis D The number of biological replicates (n = 6) was based on previous studies and corresponds to the number of replicates generally used in cell culture RT-qPCR experiments.
Statistical methods for results significance E All biological samples (n = 6) were measured in triplicate (n = 3) and an arithmetic mean of each Cq triplett used for further analysis. The stability of each candidate was calculated with four different mathematical algorithms: geNorm, NormFinder, BestKeeper and the comparative ΔCq method. Stability calculations were done with the official Microsoft-Excel-based software applets for geNorm, NormFinder and BestKeeper according to developers' instructions. For the comparative ΔCq method manual calculations were performed. The geNorm and NormFinder algorithms require the transformation of the raw Cq data to linear scale expression quantities Q corresponding to the qPCR efficiency (E) of each gene: Q = E -(Cqmin-Cqsample) with the lowest Cq value corresponding to a quantity of 1 for each candidate reference gene. The genes were ranked according to their stability values (geNorm: M, NormFinder: ρig/σi, deltaCT: mean SD of ∆Cq; BestKeeper: Pearson's r) for each algorithm and each experimental condition as well as combined experimental conditions (no treatment + compressive force, no treatment + Agac) and a rank sum of all algorithms calculated per gene for final stability assessment with the smallest rank sum indicating the most stable reference gene. Also a pooled overall ranking for all experimental conditions was calculated. The geNorm algorithm was used to calculate the ideal number of reference genes for reliable RT-qPCR normalization. If pairwise variation (Vn/Vn+1) between two sets of reference genes with one set including an additional reference gene was ≤0.15, this additional gene was deemed unnecessary for normalization. To assess ranking variations between the algorithms, we used IBM SPSS Statistics ® 23 (IBM, Armonk, NY, USA) to create a correlation matrix of bivariate correlations (Pearson´s correlation coefficient r, normality confirmed by Shapiro-Wilk tests and histogram evaluation) of the overall pooled stability values as calculated by two respective algorithms. (see Figures 3 and  Cq or raw data submission D Raw Cq values are provided in Figure 2 and Supplementary Table 3.

qPCR target information
Gene symbol E Provided in Table 1. We based our primer design on the officially registered target gene nucleotide sequences from the NCBI Nucleotide database (GeneBank, access: http://www.ncbi.nlm.nih.gov/nuccore). Sequence accession number E Location of amplicon D Provided in Table 1.
Amplicon length E Provided in Table 1. Target amplicon sequences were chosen to range from 60 to 150 bp with a GC content of 35-65%.

5´-reverse primer-3´
(length / Tm / %GC / max. ∆G Hairpin &Self-Dimer / Self-Comp. / Self-3'-Comp.)    Figure 2. Uncropped original gel of RT-qPCR products (amplification specifity). For each candidate reference gene / primer pair we found a single fluorescent band at the expected amplicon size. bp = base pairs. Gene names see Table 1. All RT-qPCR products were run concurrently and adjacently on the same gel, which was recorded with the gel documentation system Genoplex 2 (VWR International GmbH, Darmstadt, Germany) and its software GenoCapture (version 7.01, Synoptics Ltd., Cambridge, UK -automatic exposure, exposure time 80 ms, no binning, transillumination) as secure gel data (*.sgd) and exported as TIF image, which was inverted and cropped to encompass the relevant gel area.

Primer
Supplementary Data 1. Splice variants and secondary structure analysis of amplicons and primers of the nine evaluated candidate reference genes.
GAPDH PrimerBLAST (National Center for Biotechnology Information, Bethesda MD, USA, https://www.ncbi.nlm.nih.gov/tools/primer-blast ) Supplementary Data 4. Evaluation of qPCR primer efficiency (factor-specific). Log 10 serial dilution of cDNA stock solution (1,000,000 pg RNA equivalent) was performed in triplets. From the resulting C q values a standard curve was created by linear regression.