I have a problem with my miRNA rt qPCR kits! i dont believe its contamination at this stage, is there something else going on?

11 February 2024 4 4K Report

Using a standard rt qPCR kit. When looking at non human miRNA species there is a sort of 'background level' of expression in my results when there shouldnt be - i have tried running samples of pbs, Te buffer, RNA storage solution, H20 as a negative control and they all express at around 33 Cts - i have tried opening new RT kit, new qPCR master mix, new assays/primers, new pipettes, changed all equipment, completing in a pcr hood, all new reagents and a different lab ensirely. I use bleach, RNAseaway and ethanol all the time to remove any contamination. Still no change. I dont believe its from contamination at this stage because of how 'stable' every repeat is - I tried a stabiliser also and that is always clear too, which is also evidence it is not contamination going on here. Any sort of buffer seems to express at ct 33 has any one else experienced this? Do i have my threshold wrong on my qPCR machine? is there something going on with the sequence binding? Please help ! any advice appreciated.

Veenavadhini Ramalingam

HI,

It seems your kit seems to be fine. I am just wondering whether the thermal cycle has been calibrated (Background calibration). This calibration allows the software to eliminate background signals from the fluorescent samples, thus increasing the instrument’s precision. I am not sure which instrument you are using. But make sure all the calibrations are done regularly.

Volodymyr Dzhyvak

Danielle Lewis

It sounds like you've already taken a lot of steps to troubleshoot the issue with your miRNA TaqMan assays, and it's frustrating that you're still experiencing background expression. Here are some additional suggestions and considerations that may help you pinpoint the problem:

Check for primer-dimers: Sometimes, primer-dimers or non-specific amplification products can contribute to background signal. You can run a dissociation curve analysis after your qPCR to check for the presence of primer-dimers or other non-specific products. If present, you may need to optimize your PCR conditions or redesign your primers.

Evaluate PCR cycling conditions: Ensure that your PCR cycling conditions are optimized for miRNA amplification. This includes the annealing temperature, extension time, and number of cycles. Suboptimal cycling conditions can contribute to background signal or non-specific amplification.

Assess primer specificity: Double-check the specificity of your TaqMan assays by performing a BLAST search or using other sequence alignment tools. Ensure that the primers are specific to your target miRNA and do not have significant homology to other sequences.

Consider RNA degradation: Ensure that your RNA samples are of high quality and have not undergone degradation, which can lead to non-specific amplification. Use methods such as RNA integrity analysis (e.g., Bioanalyzer or TapeStation) to assess RNA quality.

Reassess qPCR threshold setting: It's possible that the threshold setting on your qPCR machine may need adjustment. Make sure that the threshold is set appropriately to accurately determine the Ct values for your samples.

Experiment with different negative controls: Try using different types of negative controls, such as RNase-free water or a no-reverse transcription control, to further assess background signal. This can help confirm whether the background signal is due to contamination or other factors.

Consult with technical support: If you're still unable to resolve the issue, consider reaching out to the technical support team of the manufacturer of your TaqMan assays. They may be able to provide additional troubleshooting advice or insights specific to their product.

By systematically evaluating these factors and considering additional possibilities, you may be able to identify the source of the background signal in your miRNA TaqMan assays and improve the reliability of your results.

John Hildyard

Really, any PCR-based reaction will tend to produce weird stuff if you leave it long enough: when the reaction is exponential, anything, anything that the primers can bind (including the taqman probe, and themselves) to will tend to end up being amplified. Even if the binding affinity is terrible and the chances are almost zero, it only needs to happen once to get something that can be amplified by subsequent rounds.

But that's usually ok: Cq of 33 is very, very close to the barest minimum quantification anyway (rule of thumb is that Cq 35 roughly corresponds to a single target molecule per well), and if your miR of interest is routinely present at Cq values comparable to your 'background' Cq of 33, then it's safe to conclude that your miR of interest isn't meaningfully present.

In my experience, miRs are usually fairly robustly expressed (Cq 20-26) or not at all (Cq 33+).

It honestly sounds to me like you're wasting a lot of time, energy and effort trying to remove non-existent contamination: this isn't contamination, it's just the way your assay works, and that's...ok: assays are rarely perfect.

Danielle Lewis

John Hildyard this is a great response and exactly where my head is.

Is it possible to extract RNA from blood using trizol extraction method?

Gel electrophoresis result for total RNA samples, 1.5 Agarose gel was used, How to improve the integrity of RNA?

No title

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

MiRNA qpcr problem?

Any specific pipeline or parameter to analyse total transcriptomics study of brain tissue samples?

Why is the efficiency of viral mRNA electroporation in PBMC low?

Can MicroRNA cross the blood brain barrier?

What is the best concentration of cDNA to be used for qPCR?

Recommendations on homogenizers for RNA isolation?

RNA-seq DEGs downstream analysis and reporting in literature?