CD74: Quantitative PCR with a hydrolysis probe and DNA binding dye

In chipPCR: Toolkit of Helper Functions to Pre-Process Amplification Data

Description

Quantitative PCR (qPCR) with a hydrolysis probe and DNA binding dye (EvaGreen) (Mao et al. 2007) was performed in the 'VideoScan' heating cooling unit. The cycle-dependent increase of the fluorescence was quantified at three different temperatures in order to estimate temperature dependent effects.

Usage

data(CD74)

Format

A data frame with 60 observations on the following 19 variables. refMFI (referenced Mean Fluorescence Intensity), fluorescence. Dilution A, 1x; Dilution B, 1:10; Dilution B, 1:100.

Cycle

PCR cycles

EG.30.A

refMFI at 30 degrees Celsius, EvaGreen, Dilution A

EG.59.5.A

refMFI at 59.5 degrees Celsius, EvaGreen, Dilution A

EG.68.5.A

refMFI at 68.5 degrees Celsius, EvaGreen, Dilution A

HP.30.A

refMFI at 30 degrees Celsius, hydrolysis probe, Dilution A

HP.59.5.A

refMFI at 59.5 degrees Celsius, hydrolysis probe, Dilution A

HP.68.5.A

refMFI at 68.5 degrees Celsius, hydrolysis probe, Dilution A

EG.30.B

refMFI at 30 degrees Celsius, EvaGreen, Dilution B

EG.59.5.B

refMFI at 59.5 degrees Celsius, EvaGreen, Dilution B

EG.68.5.B

refMFI at 68.5 degrees Celsius, EvaGreen, Dilution B

HP.30.B

refMFI at 30 degrees Celsius, hydrolysis probe, Dilution B

HP.59.5.B

refMFI at 59.5 degrees Celsius, hydrolysis probe, Dilution B

HP.68.5.B

refMFI at 68.5 degrees Celsius, hydrolysis probe, Dilution B

EG.30.C

refMFI at 30 degrees Celsius, EvaGreen, Dilution C

EG.59.5.C

refMFI at 59.5 degrees Celsius, EvaGreen, Dilution C

EG.68.5.C

refMFI at 68.5 degrees Celsius, EvaGreen, Dilution C

HP.30.C

refMFI at 30 degrees Celsius, hydrolysis probe, Dilution C

HP.59.5.C

refMFI at 59.5 degrees Celsius, hydrolysis probe, Dilution C

HP.68.5.C

refMFI at 68.5 degrees Celsius, hydrolysis probe, Dilution C

Details

The aim was to amplify MLC-2v in the 'VideoScan' platform while the intercalating dye EvaGreen and a hydrolysis probe for MLC-2v were used simultaneously. The primer sequences for MLC-2v were taken from Roediger et al. (2013). The amplification was detected in solution of the '1 HCU' (see Roediger et al. 2013 for details). A 20 micro L PCR reaction was composed of 500 nM primer (forward and reverse), 1x Maxima Probe qPCR Master Mix (Fermentas), 1 micro L template (MLC-2v amplification product in different dilutions), 50 nM hydrolysis probe probe for MLC-2v, 0.5 x EvaGreen and A. bidest. During the amplification, fluorescence was measured at 3 different temperatures, at 59.5 degrees Celsius the annealing temperature, at 68.5 degree Celsius the elongation temperature and at 30 degrees Celsius. The FAM channel was used to monitor EvaGreen and the Cy5 channel to monitor the MLC-2v specific hydrolysis probe.

Source

Claudia Deutschmann & Stefan Roediger, BTU Cottbus - Senftenberg, Senftenberg, Germany

References

A Highly Versatile Microscope Imaging Technology Platform for the Multiplex Real-Time Detection of Biomolecules and Autoimmune Antibodies. S. Roediger, P. Schierack, A. Boehm, J. Nitschke, I. Berger, U. Froemmel, C. Schmidt, M. Ruhland, I. Schimke, D. Roggenbuck, W. Lehmann and C. Schroeder. Advances in Biochemical Bioengineering/Biotechnology. 133:33–74, 2013.

Mao, F., Leung, W.-Y., Xin, X., 2007. Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnol. 7, 76.

Examples

# First example
# Comparison of smoothers and filter on amplification curves
# Amplification curves were measured at three temperature (30, 
# 59.5, 68.5 degrees Celsius) using the 'VideoScan' 'HCU' (see 
# Roediger et al. 2013 for details). MLC-2v was amplified.
# The change of fluorescence was monitored by the intercalating
# dye EvaGreen and hydrolysis probes.

data(CD74)
default.par <- par(no.readonly = TRUE)
par(mfrow = c(1,2))
plot(NA, NA, xlim = c(1,30), ylim = c(0,2), xlab = "Cycle", 
     ylab = "MFI", main = "VideoScan HCU\nRaw Data")
lim <- 1:30
for (j in c(2:4)) {
  for (i in seq(j,19,6)) {
    lines(CD74[lim, 1], CD74[lim, i], col = 1) 
  }
}
for (j in c(5:7)) {
  for (i in seq(j,19,6)) {
    lines(CD74[lim, 1], CD74[lim, i], col = 2) 
  }
}

plot(NA, NA, xlim = c(1,30), ylim = c(0,1.8), xlab = "Cycle", 
     ylab = "MFI", main = "VideoScan HCU\nSmoothed Data")
lim <- 1:30
for (j in c(2:4)) {
  for (i in seq(j,19,6)) {
    lines(CD74[lim, 1], smoother(CD74[lim, 1], CD74[lim, i], trans = TRUE), 
	  col = 1) 
  }
}
for (j in c(5:7)) {
  for (i in seq(j,19,6)) {
    lines(CD74[lim, 1], smoother(CD74[lim, 1], CD74[lim, i], trans = TRUE), 
	  col = 2) 
  }
}
par(default.par)

chipPCR documentation built on March 5, 2021, 9:06 a.m.