Azure Cielo Real-Time PCR Systems User Manual
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How do I know how many replicates I need to run?
Technical replicates are necessary to show how small differences in pipetting or template quantities may
impact analysis of the data. If replicates are consistent and show low variation from one replicate to the next,
fewer replicates will be needed.
Detection of the level of change between a control group versus an experimental group will also need to be
considered when determining an appropriate number of replicates. Larger differences will likely require fewer
replicates, with more minute differences requiring more to ensure statistical significance.
Do I need to do a standard curve if I am not trying to determine copy number or quantity?
Standard curves can provide very useful information concerning the qPCR assay even if copy number or
quantity is not the intended result. Running a standard curve over a large dilution series can clearly define
over which Ct values the assay is reliable. Evaluating the standard curve can also provide information about
the efficiency of the assay, and if optimization will be necessary.
What is a multiplex?
A multiplex reaction simply means more than one assay is run within the same sample volume (well). It is
common in probe-based analysis to see duplex or triplex reactions. The benefit to this is that more than one
gene can be evaluated within one sample aliquot, preserving valuable template.
It is important to thoroughly test the combinations of primers used within a multiplex to ensure they will work
well together, and not cross react. Good design can help minimize interactions of primers for different targets.
Why is a melt curve recommended if I use SYBR Green?
SYBR Green is a nonspecific binding dye. It will bind to any double stranded DNA molecule. If there are primer
dimer interactions, or other off target primer to template binding, SYBR Green will not distinguish between
the desired target and other products.
While adding melt curve analysis to the end of a SYBR Green run will delay the results, it will provide valuable
data as to the specificity of the SYBR Green signal that has been recorded.
If I am trying to look at gene expression, how do I know if I am seeing genomic DNA contribution in
my results?
When performing gene expression analysis, there are a few important elements to consider during planning
to help minimize or eliminate any genomic DNA contribution to your results.
Good primer designs are necessary such that only sequences related to the messenger RNA are amplified.
For Eukaryotic systems, this will involve selection of sequence that represents exon to exon junctions that
would be in messenger RNA only. Genomic DNA would contain introns, which would far exceed the base
pair size of the targeted amplicon and would likely not amplify under the conditions used for gene expression
analysis (short extension time). There are many resources available to assist with primer design. There are
also many designs available which have already been tested and proven to work.
During RNA isolation, it is common to DNase-treat at some point during the extraction, or after, to eliminate
much of the genomic DNA. Depending on the extraction method, and the type of DNase treatment, the
effective removal of the genomic DNA will vary widely.
Finally, running an no Reverse Transcriptase (RT) control when cDNA is manufactured can inform to the level,
if any, of genomic DNA contamination present. This no RT control should be run for every extraction sample
and should be prepared at the same time as the + RT samples. Including a no RT control along with any
cDNA samples in a qPCR run can show how much of the signal is due to genomic DNA.