Statistical aspects of quantitative real-time PCR experiment design.

نویسندگان

  • Robert R Kitchen
  • Mikael Kubista
  • Ales Tichopad
چکیده

Experiments using quantitative real-time PCR to test hypotheses are limited by technical and biological variability; we seek to minimise sources of confounding variability through optimum use of biological and technical replicates. The quality of an experiment design is commonly assessed by calculating its prospective power. Such calculations rely on knowledge of the expected variances of the measurements of each group of samples and the magnitude of the treatment effect; the estimation of which is often uninformed and unreliable. Here we introduce a method that exploits a small pilot study to estimate the biological and technical variances in order to improve the design of a subsequent large experiment. We measure the variance contributions at several 'levels' of the experiment design and provide a means of using this information to predict both the total variance and the prospective power of the assay. A validation of the method is provided through a variance analysis of representative genes in several bovine tissue-types. We also discuss the effect of normalisation to a reference gene in terms of the measured variance components of the gene of interest. Finally, we describe a software implementation of these methods, powerNest, that gives the user the opportunity to input data from a pilot study and interactively modify the design of the assay. The software automatically calculates expected variances, statistical power, and optimal design of the larger experiment. powerNest enables the researcher to minimise the total confounding variance and maximise prospective power for a specified maximum cost for the large study.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Selection of suitable reference genes for real-time PCR studies of early developmental stages of sturgeons

In quantitative real-time PCR, the mRNA level can be quantified in relative terms based on the expression ratio of mRNAs of the target gene and an internal reference gene. Since, an internal standard should be expressed at a constant level among different tissues of an organism at all stages of development, and should be unaffected by the experimental treatment, the stability of different refer...

متن کامل

Standardisation of data from real-time quantitative PCR methods – evaluation of outliers and comparison of calibration curves

BACKGROUND As real-time quantitative PCR (RT-QPCR) is increasingly being relied upon for the enforcement of legislation and regulations dependent upon the trace detection of DNA, focus has increased on the quality issues related to the technique. Recent work has focused on the identification of factors that contribute towards significant measurement uncertainty in the real-time quantitative PCR...

متن کامل

Evaluation of a new set of Real-Time PCR for Brucella detection within human and animal samples

A quantitative TaqMn Real-Time PCR assay was developed and its diagnostic value on human serum and livestock samples were evaluated. Brucella species could be distributed through communities as a biological agent. Rapid detection of biological threat agents is critical for timely therapeutic administration. Quantitative real-time PCR provides a rapid, sensitive and specific tool for molecular i...

متن کامل

مقایسه تاثیر مهاری ژل APF و ژل سدیم فلوراید (NAF) بر روی غلظت میکروارگانیزم های پوسیدگی زا در محیط دهان با روش Quantitative Real Time PCR

Background and purpose: Several factors are involved in caries prevention in children. One of the most effective factors is the appropriate use of fluoride. Fluoride induces its main effects in caries prevention through antibacterial effects and topical contact with enamel. In this study the inhibitory effects of sodium fluoride (NaF) and acidulated phosphate fluoride (APF) gels on cariogenic m...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:
  • Methods

دوره 50 4  شماره 

صفحات  -

تاریخ انتشار 2010