So we have been looking at the relative expression of mRNA for an endogenous opioid in 3 different brain regions of our mice using qPCR. For each brain region we want to run two replicate plates and then compare expression across both plates.
The problem is when we compared the subjects across the plates, we found that there was a great deal of variability between relative expression of the target mRNA for each subject. For example in a knockdown mouse we found that the relative expression of the mRNA increased when we compared plate 1 to plate 2.
Before we run these plates again I want to make sure I am doing everything I can to minimize any variation between the two plates.
We don't think the problem is pipetting error because when you look at the standard deviation of biological replicates it is low (we are aiming for bellow 0.2.) And we don't think it is sample degradation since expression of the knockdown went up with the second plate.
We are also using all of the same reagents for both plates.
So it should be noted we are a new lab and we have had some problems with equipment and supplies. Things we think might be going wrong:
- The tips have not been consistent across plates (we have been trying to figure out which brand of tips work best with our pipettes) and I am worried that some were not as accurate at the 1ul volume.
- The plates were run on two different machines- we have to use a core lab to run our plates so we can't always reserve the same machine each time.
Are there any other variables we haven't thought about that could be affecting this?
Hi Amanda Harmon ,
Several other potential factors that could affect are:
-Operator error: even the most skilled scientists make mistakes now and again.
-Instrument error:this will be most obvious in assays used to analyse several gene targets. Unusual patterns may be noted such that there is a positional relationship between affected sample wells. Sudden spikes in data usually indicate instrument issues.
-Reagent batch failure/incorrect reagents used for the instrument or assay (e.g. SYBR®Green I reagents with a probe assay).
-Template inappropriately checked for quality (i.e. incorrect concentration, inhibitors present,template degraded).
-Probe label may be photobleached: due to poor manufacture or incorrect storage;
-Inappropriate Tm for primers.
-Inappropriate primer concentrations.
-Incorrect or inappropriate sequences (e.g. gDNA or cDNA specific assay used for the wrong template).
-Contamination.
-Inappropriate PCR profile executed for the reagents used, e.g. antibody-inactivated enzymes need very short activation times and are less efficient if longer times are used and vice versa. A chemically modified enzyme must have an adequate activation step or else the assay will fail completely.
*Other than that, make sure to determine the integrity and purity of nucleic acid samples. Take note that the presence of inhibitors differentially affects qPCR assays variability. Within the context of qPCR assays, the significant forms of contamination refer to nucleic acid template present in the sample which may be detected along with the specific target, thus generating a false positive, or material that may inhibit downstream reactions resulting in a reduced or failed reaction (false negative).
*PCR assays are especially vulnerable to contamination with the specific target sequence of interest
*When analysing RNA, it is important to ensure there is no genomic DNA contamination (gDNA), especially if the assays in use do not discriminate between the gDNA and cDNA sequences. Enzymatic treatment of the samples with DNase I is recommended to remove contaminating gDNA, and is particularly important for investigations of gene expression.
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