A Novel Method to Compensate for Different Amplification Efficiencies between Patient DNA Samples in Quantitative Real-Time PCR

Abstract

Quantification of residual disease by real-time polymerase chain reaction (PCR) will become a pivotal tool in the development of patient-directed therapy. In recent years, various protocols to quantify minimal residual disease in leukemia or lymphoma patients have been developed. These assays assume that PCR efficiencies are equal for all samples. Determining t(14;18) and albumin reaction efficiencies for sixteen follicular lymphoma patient samples revealed higher efficiencies for blood samples than for lymph node samples in general. However, within one sample both reactions had equivalent efficiencies. Differences in amplification efficiencies between patient samples (low efficiencies) and the calibrator in quantitative analyses result in the underestimation of residual disease in patient samples whereby the weakest positive patient samples are at highest error. Based on these findings for patient samples, the efficiency compensation control was developed. This control includes two reference reactions in a multiplex setting, specific for the beta-actin and albumin housekeeping genes that are present in a constant ratio within DNA templates. The difference in threshold cycle values for both reference reactions, ie, the Ct(2) value, is dependent on the amplification efficiency, and is used to compensate for efficiency differences between patient samples and the calibrator. The beta-actin reference reaction is also used to normalize for DNA input. Furthermore, the efficiency compensation control facilitates identification of patient samples that are so contaminated with PCR inhibitory compounds that different amplification reactions are affected to a different extent. Accurate quantitation of residual disease in these samples is therefore impossible with the current quantitative real-time PCR protocols. Identification and exclusion of these inadequate samples will be of utmost importance in quantitative retrospective studies, but even more so, in future molecular diagnostic analyses.

HDAC is essential for epigenetic regulation of Thy-1 gene expression during LPS/TLR4-mediated proliferation of lung fibroblasts

“…Amplification was performed on the IQ5 PCR System (Bio-Rad, USA) with an initial denaturing step at 95°C for 15 s, 45 cycles of denaturing at 95°C for 5 s, and annealing at 60°C for 30 s. Thy-1 gene expression, which was normalized to GAPDH expression, was analyzed by the ΔΔCt method. 13 Western Blot Analysis Cells were lysed on ice with RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride (PMSF), 20 μg/ml aprotinin, 20 μg/ml leupeptin, 10 μg/ml pepstanin A, and 150 mM benzamidine) for 15 min. Supernatants were collected after centrifugation and protein quantification was determined using the BCA method.…”

The prolipoprotein diacylglyceryl transferase (Lgt) of Enterococcus faecalis contributes to virulence

“…Amplification (using 5 ml of a 1 : 100 cDNA dilution), detection (with automatic calculation of the threshold value), and real-time analysis were performed twice for each cDNA sample using the iCycler iQ Detection System (Bio-Rad Laboratories). Relative mRNA levels for each gene in each sample were calculated using comparative cycle time, as described elsewhere (Meijerink et al, 2001).…”

EVI1 overexpression in distinct subtypes of pediatric acute myeloid leukemia

“…The relative expression of the EVI1 transcripts (EVI1-1A, -1B, -1D, and -3L) and MDS1/EVI1 transcript was calculated using the comparative cycle time (DCt) method, with GAPDH as the housekeeping gene. 32 Primer en probe sequences are shown in Supplementary Table 1. A sample was considered EVI1 þ with RQ-PCR, if the cumulative relative expression EVI1-1A, -1B, and -3L to GAPDH was above 1.5%.…”