RQ_ddCt {RQdeltaCT}

RQ_ddCt

Description

This function performs relative quantification of gene expression using 2^-ddCt method.

Usage

RQ_ddCt(
  data,
  group.study,
  group.ref,
  do.tests = TRUE,
  pairwise = FALSE,
  alternative = "two.sided",
  p.adjust.method = "BH",
  save.to.txt = FALSE,
  name.txt = "ddCt_RQ_results"
)

Arguments

data

Data object returned from delta_Ct() function.

group.study

Character: name of study group (group of interest).

group.ref

Character: name of reference group.

do.tests

Logical: if TRUE, statistical significance of delta delta Ct values between compared groups is calculated using Student's t test and Mann-Whitney U test. Default to TRUE.

pairwise

Logical: set to TRUE if a pairwise analysis should be done.

alternative

Character: alternative hypothesis, must be one of "two.sided" (default), "greater" or "less".

p.adjust.method

Character: p value correction method for multiple testing, one of the "holm", "hochberg", "hommel", "bonferroni", "BH" (default), "BY","fdr", or "none". See documentation for stats::p.adjust() function for details.

save.to.txt

Logical: if TRUE, returned table with results is saved to .txt file. Default to FALSE.

name.txt

Character: name of saved .txt file, without ".txt" name of extension. Default to "results_ddCt".

Details

This function performs: 1. Calculation of means (returned in columns with the "_mean" pattern) and standard deviations (returned in columns with the "_sd" pattern) of delta Ct values for analyzed genes across compared groups. 2. Normality tests (Shapiro_Wilk test) of delta Ct values of analyzed genes across compared groups and returned p values are stored in columns with the "_norm_p" pattern. 3. Calculation of differences in mean delta Ct values of genes between compared groups, obtaining delta delta Ct values (returned in"ddCt" column). 4. Calculation of fold change values (returned in "FCh" column) for each gene by exponentiation of ddCt values using 2^-ddCt formula. 5. Statistical testing of differences between study group and reference group using Student's t test and Mann-Whitney U test. Resulted statistics (in column with "_test_stat" pattern) and p values (in column with "_test_p" pattern) are returned. 6. P values adjusted for multiple testing using a selected method.

Value

Data frame with relative quantification results.

Examples

library(tidyverse)
library(coin)
data(data.Ct)
data.CtF <- filter_Ct(data.Ct,
                      remove.Gene = c("Gene2","Gene5","Gene6","Gene9","Gene11"),
                      remove.Sample = c("Control08","Control16","Control22"))
data.CtF.ready <- make_Ct_ready(data.CtF, imput.by.mean.within.groups = TRUE)
data.dCt <- delta_Ct(data.CtF.ready, ref = "Gene8")
results.ddCt <- RQ_ddCt(data.dCt, "Disease", "Control")
head(results.ddCt)