Validation of the 2-????Ct Calculation as an Alternate Method of Data Analysis for Quantitative PCR of BCR-ABL P210 Transcripts

Abstract

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder that is characterized by the presence of a reciprocal translocation between chromosomes 9 and 22 and results in the formation of the Philadelphia (Ph1) chromosome and is present in most of CML patients. The Ph1 chromosome forms a chimeric gene that encodes an abnormal P210 mRNA transcript in most CML patients. Surveillance for minimal residual disease by detection of BCR/ABL transcripts is currently done mostly by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Quantitation of BCR/ABL transcripts can monitor tumor load and the outcome of therapy. Absolute quantification determines the input copy number of the transcript of interest, usually by plotting the amount of PCR product onto a standard curve based on serial dilutions of the same product cloned in plasmids. Relative quantification describes the change in expression of the target gene in the patient sample relative to that of a control transcript by using the 2-DeltaDeltaCt calculation. The results of real-time RT-PCR for BCR/ABL transcripts are often analyzed by using plasmid DNA standard curves. In the present study, 79 BCR/ABL transcript-positive samples from CML patients who were being monitored for minimal residual disease by real-time quantitative RT-PCR were studied to determine whether the 2-DeltaDeltaCt approach was equivalent to the plasmid standard curve method. BCR/ABL P210 transcripts were quantitated using both the plasmid standard curve method and the 2-DeltaDeltaCt calculation. The comparison of both methods revealed a highly significant and linear correlation between the plasmid standard curve method and the 2-DeltaDeltaCt calculation (R2=0.98, P<0.0001). Furthermore, there was a reduction of preparation time, contamination risk, and reagent usage. The 2-DeltaDeltaCt calculation is a convenient alternative method to derive accurate quantitative information from real time PCR assays.

Microarray pathway analysis indicated that mitogen-activated protein kinase/extracellular signal-regulated kinase and insulin growth factor 1 signaling pathways were inhibited by small interfering RNA against AT‑rich interactive domain 1A in endometrial cancer

“…The thermocycling conditions were as follows: Degeneration at 95˚C for 30 sec, annealing at 60˚C for 30 sec and extension at 72˚C for 1 min, for 40 cycles. Relative gene expression levels were calculated using the 2 -ΔΔcq method (12) and were normalized to the expression of GAPDH (forward, 5'-TGA CTT CAA CAG CGA CAC CCA-3' and reverse, 5'-CAC CCT GTT GCT GTA GCC AAA).…”

De novo ceramide synthesis is involved in acute inflammation during labor

“…The amplification of target genes (IL-6, TNF-α, SPTLC 1, SPTLC 2, SPTLC 3: primer sequences as previously reported (Hornemann et al, 2006;Dechecchi et al, 2011) was performed using the Syber Green system (Qiagen, Milan, Italy). Relative mRNA expression of target genes was normalized to the endogenous GAPDH control gene and results calculated by 2 -ΔΔCt method (Arocho et al, 2006), as labored vs. control cesarean placentas. RT-PCR determinations were performed in triplicate.…”

Effects of sexual steroids on the expression of foxl2 in Gobiocypris rarus

“…The relative expression of foxl2 in samples was calibrated/normalized against β-actin by using the 2 − ΔΔCt calculation method (Arocho et al, 2006):…”