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Home > Products and Services > PCR Home > Custom PCR Array
Custom PCR Array
Custom PCR Arrays Tailored to Your Research
Custom Profiler RT² PCR Arrays for gene expression analysis are made with RT² Primer Assays, which are all wet-bench laboratory verified (with a performance guarantee).
Pathways available for other species 
If you like the content for any of our pathways, but are working on a
different species than those mentioned above, please contact us at Support@SABiosciences.com
to inquire about a Species Conversion for this pathway to support your species
of interest.
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Click for layout options
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Options for Customizing PCR Arrays
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Modify Catalogued PCR Arrays: Add Your
Favorite Genes (Up to 4) to Existing PCR Array Gene Lists
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Build Your Own Custom PCR Arrays: Provide Your
Own List of Genes
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PCR Array Plate Format
How to Order:
- Select a plate layout to match the gene number and the number of samples
you would like to analyze.
- Please have the type of RT-PCR instrument available when placing the order.
- Determine the number and types of plates required.
- Use the following Excel file to enter your gene list.
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Contact SABiosciences Technical Support at 1-888-503-3187 to submit your gene
list and obtain a quote.
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Quality Assurance / Quality Control
(QA/QC) Parameters
Primer Quality Control (QC)
Any algorithm's primer design must also be experimentally validated for
high-performance with wet bench quality control protocols starting with two
major success criteria.
- First, a melt curve analysis must verify that a single gene-specific
product is produced. Following the melt curve, an agarose gel can also
be run to further verify a single product of the predicted size, based
on the amplicon design, without primer dimers or off-target
amplifications.
- Second, the amplification efficiency must be greater than 90 percent
for accurate and reliable results. If a real-time RT-PCR assay does not
meet all of the above requirements, then the quality control fails, and
the assay must be re-designed.
QA/QC Specificity
All real-time PCR assays must generate a single band of the correct size
for the results to accurately represent the expression of the queried gene.
Secondary products confound the analysis. If using SYBR Green-based
detection, you can tell if your real-time PCR assays are specific enough by
simply running the default melting program on your instrument immediately
after the completion of the cycling program. A single peak indicates a
single melting event, and therefore a single product.
| Figure 1: |
Human XpressRef Universal Total RNA was
characterized on the Human Common Cytokines (B) RT² Profiler™ PCR Arrays,
followed by dissociation (melt) curve and gel electrophoretic analyses. The RT²
qPCR™ Assay specifically detects an individual gene, in particular for
the displayed cytokine genes, whose specific assays tend to be notoriously
difficult to design.
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Efficiency
Of the various methods of determining amplification efficiency, the most
rigorous and classical method examines the slope of a calibration curve,
much like those used to assess dynamic range. An assay with 100 percent
efficiency yields a -3.33 calibration curve slope, a relatively narrow
standard deviation about that mean.
| Figure 2: |
A representative set of assays for 4,000 genes used in the RT² PCR
Arrays demonstrate their average amplification efficiency of 99% and their
95% confidence interval about the mean from 90-110%. Consistently high
amplification efficiencies enable PCR Arrays to accurately analyze multiple
genes simultaneously using the ΔΔCt method. |
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Primer Design Algorithm
Primer design algorithm is key to effective qPCR based gene expression
analysis. Designs must meet several important thermodynamic and sequence
criteria. Primers are designed such that they must detect every alternative
transcript and splicing variant of the queried gene so as not to miss any
genes.
To do so, all known entries in the public databases should be found and
aligned to reveal a common gene-specific region for primer design.
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By controlling the GC content, primer length, and the
primer melting temperature range, each assay can use a standard set of
PCR cycling conditions.
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Uniform cycling conditions, in turn, allow researchers
to scale up from a single assay, to multiple assays on an entire 96- or
even 384-well plate.
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Single Nucleotide Polymorphism (SNP) analysis can
eliminate repetitive sequences so that any individual source of total
RNA may be analyzed with the same assay.
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BLAST analysis further insures that the chosen primer
sequences are sufficiently different from the rest of the transcriptome
in the species of interest.
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Stability at the 3'-end of the primers controls the
start position for the DNA polymerase, further enhancing specificity.
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System Performance
Sensitivity and Dynamic Range
Sensitivity may also be judged by how many copies of cDNA can be
detected. When analyzing the expression of multiple genes at once in the
same cycling run, some genes may be expressed at very low copy numbers while
other may be expressed at much higher copy numbers. Real-time PCR assays, in
general, have the unique potential capability of detecting transcripts down
to one individual copy and up to several orders of magnitude more.
Researchers have come to expect or even assume that real-time PCR assays
indeed detect a wide variety of transcripts expressed at very different
levels.
Reproducibility
Researchers are also understandably concerned about the reproducibility
of their real-time PCR assay results, if only to help make sure that
reviewers will let their results be published. In addition, profiling
multiple genes in the same sample also requires a high degree of
reproducibility. When looking at enough genes to fill an entire 96-well
or even 384-well plate, the assays must be reproducible enough so that the
data can be legitimately compared between separate and individual runs,
plates, and samples
The MAQC brain reference RNA sample4,5 was reverse
transcribed and run on four replicate Human
Drug Metabolism PCR Arrays three months apart by two different investigators
each using a different
production lot. The raw data from each end-user's four replicates with all
four of the other enduser's
replicates in a scatter plot, and fit to a straight line with a slope of
one. The average Ct value
correlation coefficient between the replicate runs by the respective
end-users was 0.995 +0.001 and
0.998 +0.000.
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How PCR Array Control Elements Work
Many factors are known to influence real-time PCR assay analyses, even
if the performance of the assay itself is validated and optimized-from
input RNA quality, RT condition, the PCR cycling conditions, and to
the performance of real-time PCR instruments. For data normalization
purposes, we recommend our customers include a set of control elements
into each custom-made PCR array for Biomarker Validation array.
As a company which provides the reagents and pre-validated assays
to researchers, we have come across many things that could go astray
in a qRT-PCR assay. Problems in the quality of RNA encompasses the
majority (>90%) of the cases. In addition, instrument-related
problems such as suboptimal performance of the real-time PCR
instrument, high background contamination on the thermal block, or
incorrect PCR program setup, which will greatly affect the success of
PCR, often go unnoticed or ignored. Hence, the possibility of many
unforeseen variables that could occur during the qRT-PCR processes
necessitates the inclusion of controls that can monitor the outcomes
of each step of qRT-PCR.
To have reliable and reproducible qRT-PCR assays,
three requirements have to be met. These include:
- Minimal genomic DNA contamination.
- Intact RNA free of any impurities that inhibit reverse
transcription or PCR
- Correct PCR cycling conditions
To serve these purposes, we have developed a proprietary panel of
controls which would give researchers confidence in their qRT-PCR
results. Furthermore, these controls would greatly facilitate
researchers and save their time in the troubleshooting processes,
should a problem arise in their qRT-PCR assays.
- Genomic DNA Contamination (GDC) control: detects a specific
genomic DNA sequence within an ORF-free intergenic region
- Reverse Transcription Control (RTC): detects an artificial
External RNA Control sequence spiked into the first strand cDNA
synthesis reaction
- Positive PCR Control (PPC): Detects a pre-dispensed external DNA
template of known copies to produce a defined Ct value under
proper PCR conditions
Minimal genomic DNA contamination
So why do we care about genomic DNA contamination? This is because
genomic DNA found in RNA preparations can act as an effective template
during PCR, resulting in undesirable false positive signals unrelated
to mRNA. Strategies such as cross-intron primer design may still not
be able to avoid amplification from the contaminating genomic DNA due
to presence of large number of processed pseudogenes in the mammalian
genomes.
The GDC assay can be used to evaluate how effective genomic DNA
contamination is removed from RNA samples by DNAse treatment.
Traditionally, researchers perform qPCR of RT-minus assays (also known
as no reverse transcription (NRT) assays) to monitor the level of
genomic DNA contamination in their samples. However, this would mean
performing an additional first strand synthesis reaction and PCR assay
for each targeted gene.
| Figure 6: |
RNA from HEK 293T cells, mouse spinal tissue, mouse brain tissue,
or rat brain tissue was characterized on PCR Arrays before (purple
bars) and after (yellow bars) treatment with gDNA Elimination Buffer
from the RT² First Strand Kit (330401). Successful removal of genomic DNA
contamination is indicated by GDC Ct values greater than 35.
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Intact RNA free of any impurities that inhibit reverse
transcription or PCR
| Figure 7: |
Tissue contaminants and lysis or wash reagent
impurities inadvertently co-purifying with RNA during isolation
adversely affect RNA integrity or inhibit reverse transcriptase
activity. Human universal RNA was characterized without or with a
small amount of TRIZOL® reagent or treatment with a magnesium salt to
simulate RNA degradation. Contaminated or degraded RNA resulted in
higher threshold cycle (Ct) values for both the genes of interest and
the RTC, and increased the Ct difference between the RTC and PPC
controls above the pass/fail threshold of five (5) cycles, indicating
that reverse transcription was impaired. |
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Correct PCR cycling conditions
Real-time PCR instruments, especially in core facilities, are often
heavily used and their performance will deteriorate over time. If
these instruments are not properly maintained, data generated from
them may not be reliable. The PPC can be used to assess the
performance of real-time PCR instruments and to determine if they need
to be serviced.
| Figure 8: |
Incorrect setup of PCR cycling conditions or the
presence of PCR inhibitors adversely affects qRT-PCR results.
Universal RNA was characterized on PCR Arrays with or without the
ten-minute 95 ºC heat activation step required to begin the
cycling program. The correct program yielded the predicted PPC Ct
value of 20, while the wrong program failed to adequately amplify the
PPC template (Ct = 36).
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Data Analysis
A technical support representative will email you customized data
analysis template to suit your gene set after your order is placed.
Your system tool will be ready to go when your custom arrays arrive at
your lab. Please visit the RT² PCR Array Data Analysis page to learn more.
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Complete Array List
