Real-time PCR is becoming the method of choice for precise quantification of minute amounts of nucleic acids. For proper comparison of samples, almost all quantification methods assume similar PCR efficiencies in the exponential phase of the reaction. However, inhibition of PCR is common when working with biological samples and may invalidate the assumed similarity of PCR efficiencies. Here we present a statistical method, Kinetic Outlier Detection (KOD), to detect samples with dissimilar efficiencies. KOD is based on a comparison of PCR efficiency, estimated from the amplification curve of a test sample, with the mean PCR efficiency of samples in a training set. KOD is demonstrated and validated on samples with the same initial number of template molecules, where PCR is inhibited to various degrees by elevated concentrations of dNTP; and in detection of cDNA samples with an aberrant ratio of two genes. Translating the dissimilarity in efficiency to quantity, KOD identifies outliers that differ by 1.3-1.9-fold in their quantity from normal samples with a P-value of 0.05. This precision is higher than the minimal 2-fold difference in number of DNA molecules that real-time PCR usually aims to detect. Thus, KOD may be a useful tool for outlier detection in real-time PCR.

GnRH, the main regulator of reproduction, is produced in a variety of tissues outside of the hypothalamus, its main site of synthesis and release. We aimed to determine whether GnRH produced in the female rat pituitary and ovaries is involved in the processes leading to ovulation. We studied the expression patterns of GnRH and GnRH receptor (GnRH-R) in the same animals throughout the estrous cycle using real-time PCR. Hypothalamic levels of GnRH mRNA were highest at 1700 h on proestrus, preceding the preovulatory LH surge. No significant changes in the level of hypothalamic GnRH-R mRNA were detected, although fluctuations during the day of proestrus are evident. High pituitary GnRH mRNA was detected during the day of estrus, in the morning of diestrus 1, and at noon on proestrus. Pituitary GnRH-R displayed a similar pattern of expression, except on estrus, when its mRNA levels declined. Ovarian GnRH mRNA levels increased in the morning of diestrus 1 and early afternoon of proestrus. Here, too, GnRH-R displayed a somewhat similar pattern of expression to that of its ligand. To the best of our knowledge, this is the first demonstration of a GnRH expression pattern in the pituitary and ovary of any species. The different timings of the GnRH peaks in the three tissues imply differential tissue-specific regulation. We believe that the GnRH produced in the anterior pituitary and ovary could play a physiological role in the preparation of these organs for the midcycle gonadotropin surge and ovulation, respectively, possibly via local GnRH-gonadotropin axes.

Quantitative real-time PCR (qPCR) is the method of choice for specific and sensitive quantification of nucleic acids. However, data validation is still a major issue, partially due to the complex effect of PCR inhibition on the results. If undetected PCR inhibition may severely impair the accuracy and sensitivity of results. PCR inhibition is addressed by prevention, detection and correction of PCR results. Recently, a new family of computational methods for the detection of PCR inhibition called kinetics outlier detection (KOD) emerged. KOD methods are based on comparison of one or a few kinetic parameters describing a test reaction to those describing a set of reference reactions. Modern KOD can detect PCR inhibition reflected by shift of the amplification curve by merely half a cycle with specificity and sensitivity >90%. Based solely on data analysis, these tools complement measures to improve and control pre-analytics. KOD methods do not require labor and materials, do not affect the reaction accuracy and sensitivity and they can be automated for fast and reliable quantification. This review describes the background of KOD methods, their principles, assumptions, strengths and limitations. Finally, the review provides recommendations how to use KOD and how to evaluate its performance.

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