deltaCt: Perform normalization with a given housekeeping gene

In NormqPCR: Functions for normalisation of RT-qPCR data

Description

Normalise qPCR eset using a given housekeeping gene as control, then perform differential expression analysis using the delta delta Ct method

Usage

deltaCt(qPCRBatch, ...)
## S4 method for signature 'qPCRBatch'
deltaCt(qPCRBatch, hkgs, combineHkgs=FALSE, calc="arith")
deltaCq(qPCRBatch, hkgs, combineHkgs=FALSE, calc="arith")

Arguments

qPCRBatch	qPCR-specific expression set, containing qPCR data.
...	Extra arguments, detailed below
hkgs	String containing the name of the name of the housekeeping gene which will be used to normalise the rest of the genes.
combineHkgs	Logical - if TRUE, then as long as more than one housekeeper given for argument hkgs, it will combine the housekeepers by finding the geometric mean. Housekeepers can be found using geNorm or NormFinder algorithms.
calc	use arithmetic or geometric mean.

Details

Takes expression set of qPCR values and normalises them using a housekeeping gene. Returns a qPCRBatch with exprs set of the same dimensions but with the given hkg value subtracted.

Value

qPCRBatch with exprs set of the same dimensions but with the given hkg value subtracted.

Author(s)

James Perkins [e-mail]

References

Kenneth Livak, Thomase Schmittgen (2001). Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2^DDCt Method. Methods 25, 402-408, 2001 http://www.ncbi.nlm.nih.gov/pubmed/11846609

Perkins, JR, Dawes, JM, McMahon, SB, Bennett, DL, Orengo, C, Kohl, M (2012). ReadqPCR and NormqPCR: R packages for the reading, quality checking and normalisation of RT-qPCR quantification cycle (Cq) data. BMC Genomics, 13, 1:296.

See Also

selectHKs, deltaDeltaCq

Examples

  path <- system.file("exData", package = "NormqPCR")
  taqman.example <- file.path(path, "example.txt")
  qPCRBatch.taqman <- read.taqman(taqman.example)
  hkgs<-"Actb-Rn00667869_m1"
  qPCRBatch.norm <- deltaCq(qPCRBatch =  qPCRBatch.taqman, hkgs = hkgs, calc="arith")
  head(exprs(qPCRBatch.norm))

Example output

Loading required package: RColorBrewer
Loading required package: Biobase
Loading required package: BiocGenerics
Loading required package: parallel

Attaching package: 'BiocGenerics'

The following objects are masked from 'package:parallel':

    clusterApply, clusterApplyLB, clusterCall, clusterEvalQ,
    clusterExport, clusterMap, parApply, parCapply, parLapply,
    parLapplyLB, parRapply, parSapply, parSapplyLB

The following objects are masked from 'package:stats':

    IQR, mad, sd, var, xtabs

The following objects are masked from 'package:base':

    Filter, Find, Map, Position, Reduce, anyDuplicated, append,
    as.data.frame, cbind, colMeans, colSums, colnames, do.call,
    duplicated, eval, evalq, get, grep, grepl, intersect, is.unsorted,
    lapply, lengths, mapply, match, mget, order, paste, pmax, pmax.int,
    pmin, pmin.int, rank, rbind, rowMeans, rowSums, rownames, sapply,
    setdiff, sort, table, tapply, union, unique, unsplit, which,
    which.max, which.min

Welcome to Bioconductor

    Vignettes contain introductory material; view with
    'browseVignettes()'. To cite Bioconductor, see
    'citation("Biobase")', and for packages 'citation("pkgname")'.

Loading required package: ReadqPCR
Loading required package: affy
Loading required package: qpcR
Loading required package: MASS
Loading required package: minpack.lm
Loading required package: rgl
Loading required package: robustbase

Attaching package: 'robustbase'

The following object is masked from 'package:Biobase':

    rowMedians

Loading required package: Matrix
Warning messages:
1: In read.dcf(con) :
  URL 'http://bioconductor.org/BiocInstaller.dcf': status was 'Couldn't resolve host name'
2: In rgl.init(initValue, onlyNULL) : RGL: unable to open X11 display
3: 'rgl_init' failed, running with rgl.useNULL = TRUE 
4: .onUnload failed in unloadNamespace() for 'rgl', details:
  call: fun(...)
  error: object 'rgl_quit' not found 
Warning message:
In read.taqman(taqman.example) :
  Incompatible phenoData object. Created a new one using sample name data derived from raw data.

                     fp1.day3.v fp2.day3.v fp5.day3.mia fp6.day3.mia
Actb.Rn00667869_m1     0.000000   0.000000     0.000000     0.000000
Adipoq.Rn00595250_m1   0.016052  -0.116520     2.933523     2.540987
Adrbk1.Rn00562822_m1         NA         NA     6.566628     6.642561
Agtrl1.Rn00580252_s1   4.899380   5.035841     6.397364     5.680837
Alpl.Rn00564931_m1    12.531942  11.808657    13.035166    12.239549
B2m.Rn00560865_m1      0.741558   0.890717     2.040470     2.234605
                     fp.3.day.3.v fp.4.day.3.v fp.7.day.3.mia fp.8.day.3.mia
Actb.Rn00667869_m1       0.000000     0.000000       0.000000       0.000000
Adipoq.Rn00595250_m1    -0.178971    -0.563263       2.458509       2.736475
Adrbk1.Rn00562822_m1           NA           NA       3.737100       6.873568
Agtrl1.Rn00580252_s1     5.220796     4.425364       4.794776       5.345202
Alpl.Rn00564931_m1      12.394802    11.772896      12.110000      12.255186
B2m.Rn00560865_m1        0.505516     0.877598       1.927563       1.903269

NormqPCR documentation built on Nov. 8, 2020, 6:37 p.m.