rtpcr (version 2.1.2)

WILCOX_DDCt: Delta Delta Ct method wilcox.test analysis

Description

The WILCOX_DDCt function performs fold change expression analysis based on the \(\Delta \Delta C_T\) method using wilcox.test. It supports analysis of one or more target genes evaluated under two experimental conditions (e.g. control vs treatment).

Usage

WILCOX_DDCt(
  x,
  numberOfrefGenes,
  Factor.level.order = NULL,
  paired = FALSE,
  p.adj = "none",
  order = "none"
)

Value

A table containing RE values, log2FC, p-values, significance, confidence intervals, standard errors, and lower/upper SE limits.

Arguments

x

A data frame containing experimental conditions, biological replicates, and amplification efficiency and Ct values for target and reference genes. The number of biological replicates must be equal across genes. If this is not true, or there are NA values use ANODA_DDCt function for independent samples or REPEATED_DDCt for paired samples. See the package vignette for details on the required data structure.

numberOfrefGenes

Integer specifying the number of reference genes used for normalization.

Factor.level.order

Optional character vector specifying the order of factor levels. If NULL, the first level of the factor column is used as the calibrator.

paired

Logical; if TRUE, a paired wilcox.test is performed.

p.adj

Method for p-value adjustment. One of "holm", "hochberg", "hommel", "bonferroni", "BH", "BY", "fdr", or "none". See p.adjust.

order

Optional character vector specifying the order of genes in the output plot.

Author

Ghader Mirzaghaderi

Details

Relative expression values are computed using reference gene(s) for normalization. Both paired and unpaired experimental designs are supported.

Paired samples in quantitative PCR refer to measurements collected from the same individuals under two different conditions (e.g. before vs after treatment), whereas unpaired samples originate from different individuals in each condition. Paired designs allow within-individual comparisons and typically reduce inter-individual variability.

The function returns expression table. The expression table returned by `TTEST_DDCt()`, `WILCOX_DDCt()`, `ANOVA_DDCt()`, `ANCOVA_DDCt()`, and `REPEATED_DDCt()` functions include these columns: gene (name of target genes), contrast (calibrator level and contrasts for which the relative expression is computed), RE (relative expression or fold change), log2FC (log(2) of relative expression or fold change), pvalue, sig (per-gene significance), LCL (95% lower confidence level), UCL (95% upper confidence level), se (standard error of mean calculated from the weighted delta Ct values of each of the main factor levels), Lower.se.RE (The lower limit error bar for RE which is 2^(log2(RE) - se)), Upper.se.RE (The upper limit error bar for RE which is 2^(log2(RE) + se)), Lower.se.log2FC (The lower limit error bar for log2 RE), and Upper.se.log2FC (The upper limit error bar for log2 RE)

References

Yuan, J. S., Reed, A., Chen, F., and Stewart, N. (2006). Statistical Analysis of Real-Time PCR Data. BMC Bioinformatics, 7, 85.

Examples

# Example data structure
data <- read.csv(system.file("extdata", "data_Yuan2006PMCBioinf.csv", package = "rtpcr"))

# Unpaired t-test
WILCOX_DDCt(
  data,
  paired = FALSE,
  numberOfrefGenes = 1)


# Two reference genes
data2 <- read.csv(system.file("extdata", "data_1factor_Two_ref.csv", package = "rtpcr"))
WILCOX_DDCt(
  data2,
  numberOfrefGenes = 2,
  p.adj = "none")