ExProfile™ Pathway Gene qPCR Arrays profile the expression of pathway-related genes, which are carefully chosen for their close pathway correlation based on a thorough literature search of peer-reviewed publications. In each 96-well catalog array plate there are up to 84 pairs of All-in-One™ qPCR primers, which have been pre-validated and deposited in designated wells. In each plate there also are 12 wells of controls for monitoring the efficiency of the entire experimental process: from reverse transcription to qPCR reaction.
GeneCopoeia offers 96-well and 384-well custom array types and each array type has 6 layouts to choose from depending on the number of genes and replicates to be analyzed. Different controls can also be included in the arrays to help monitor the sample quality, RT and PCR reaction efficiencies and replicates reproducibility.
Genomic DNA control (GDC): detects genomic DNA contamination.
Spike-in reverse transcription control (RT): monitors the efficiency of the RT reaction.
Positive PCR control (PCR): verifies the PCR efficiency.
Housekeeping genes (HK): can be used as endogenous positive controls and for array normalization.
The All-in-One™ qPCR human- mouse- or rat-specific primers are designed by a proprietary algorithm and validated† for precision performance. Primer validation includes melting curve to ensure amplification of the correct target DNA (Figure 2). When used in combination with the All-in-One™ SYBR® Green qPCR Mix, the All-in-One™ primers deliver reliable and reproducible high performance in quantitative PCR assays.
Amplification curves
Melting curves
Agarose Gel Electrophoresis Validation Result
Figure 2. Fourty-five pairs of gene-specific All-in-One™ qPCR primers were experimentally validated to yield a single dissociation curve peak and to generate a single amplification of the correct size for the targeted genes. A cDNA pool, containing reverse transcribed products of total RNA from 10 different human tissue (lung, liver, testicle, ovary, spleen, brain, placenta, pancreas, heart and mammary), was used as the qPCR validation template. qPCR was performed using 0.2 µM primer with 2X All-in-One qPCR Mix. Reactions were incubated for 10 min. at 95°C, followed by 40 cycles of 95°C for 10 sec.; 60°C, 20 sec. and 72°C, 15 sec. using Bio-Rad iQ5 Instrument. At the end of the last cycle, the temperature was increased from 72 to 95°C to produce a melting curve. Amplification curves, melting curves and agarose gel electrophoresis (validation result for positive in odd-lane and no template control (NTC) in even lane) shown for the 10 samples.
* Non-specific amplification and absence of primer-dimers are ensured when All-in-One validated PCR primers and PCR mix are used together.
† Primers validated automatically for human, mouse and rat. Inquire for validation of other species.
