Total RNA extracted from cells was used as a template for cDNA transcription using SuperScript® III First-Strand Synthesis SuperMix for RT-qPCR (Life Technologies Corporation Cat.# 18080-400) (2), (3). The protocol formulation can be used to quantify fewer than 10 copies of a target gene using RT-qPCR, and has a broad dynamic range that supports accurate quantification of high-copy mRNA in up to 1μg of total RNA.
Master mix for cDNA synthesis:
- 2X RT Reaction Mix 10μl
- RT Enzyme Mix 2μl
- RNA (up to 1 μg) xμl
- DEPC-treated water to 20μl
Treatment with E. coli RNase H in a final volume of 50μl was used to remove the RNA template from the cDNA:RNA hybrid molecule after first-strand synthesis.
Quantitative Real-Time PCR (qPCR) Protocols and Results
To determine if the purified RNA samples could be efficiently reverse-transcribed and amplified, the samples were assayed using quantitative PCR methods without post-PCR processing. We used real-time detection of fluorescent signals during PCR cycling, significantly reducing the risk of PCR product contamination (4) , (5) . Two types of real-time qPCR instruments, iCycler IQ® Real-Time PCR Detection System (Bio-Rad Cat.# 170-8740) and Applied Biosystems 7300 Real-Time PCR instrument (LifeTechnologies, Cat.# SC7300) were used to evaluate ABL control gene copy number in each sample.
Samples (n=450; 205 BM and 245 PB) were screened on the iCycler IQ® apparatus and ABL real-time qPCR assays were performed using 2X IQ® Supermix (Bio-Rad Cat.# 170-8862) and ABL standards (Qiagen Cat.# 674691).
In addition, some samples (n=251; 138 BM and 113 PB) underwent real-time qPCR analysis for ABL detection on Applied Biosystems platform using ABL mix from ProfileQuant® kit (Qiagen Cat.# 676923) or from Alert kit (ELITechGroup, Cat.# RTSG07-210).
The standard curve was derived from three dilution points, 105–103 for Qiagen standards, as values below 103 are not acceptable for ABL, while a 102 (as in four dilution points for ELITechGroup ABL standards) dilution may be of limited use for ABL. The threshold is used to determine the threshold cycle (Cq) and is typically set in the log-linear phase of the amplification curve, where the Cq value denotes the PCR cycle at which fluorescence is detected above the background level.
Each qPCR run should conform to the following criteria:
- Slope –3.2 to –3.6
- R2 > 0.980
Runs that do not meet these criteria should be rejected.
Amplification of an endogenous control gene such as ABL is used as a reference gene to normalize target transcript levels and identify poor quality samples. ABL is the most common reference gene used due to its wide expression level across different cell types and its stability. The ability to amplify at least 10,000 ABL molecules per reaction provides, in our experience, a safety measure against poor sample quality, avoiding incorrect normalization in quantitative assay and also false-negative results. All data obtained were included in the analysis. By assessing the absolute value of the ABL housekeeping gene we estimated whether quantification results could be affected by this purification method.
qPCR results on both platforms are summarized in Figure 2 by reporting the mean ABL copy number. As shown, the housekeeping gene was detected with a value ranging from 2.59 x 104 (column 6) and 5.79 x 104 (column 3). Significant differences between the two methods were detected on peripheral blood only but still maintaining mean ABL copy number above 10,000, indicating high quality of the RNA. As shown in Figure 2, a slight difference in values obtained with the two RQ-PCR apparatus is always detected, probably due to the use of different reagents and protocols on the two instruments.