Real-Time PCR Basics Support—Troubleshooting
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No. After the addition of EDTA, there is an approximately 1:1 molar ratio of EDTA:Mg2+. EDTA chelates Mg2+ molecules on a 1:1 molar basis. Therefore, this RNA can be directly used in a reverse transcription reaction. First-strand reverse transcription buffers typically result in a final concentration of 2.5 mM Mg2+. If the reverse transcription buffer does not contain MgCl2, add it to the reaction to a final concentration of 2.25–2.5 mM.
You can run a no-RT control reaction with the SuperScript® VILO™ Master Mix by heat-inactivating the SuperScript® III Reverse Transcriptase enzyme for 5 min at 85°C. Then continue with the protocol as normal, using the mix that now has no RT activity.
It is not possible to directly measure the cDNA concentration by UV absorbance because the dNTPs from the reverse transcription step will also absorb and throw off the measurement. For an accurate reading, you would have to purify the cDNA first. Since this would lead to a loss of material, cDNA concentrations are estimated based on the initial RNA input instead. For example, if you used 100 ng of total RNA in a 20 µL reverse transcription reaction, you can assume a maximum of 100 ng of cDNA (at 5 ng/µL).
It may be possible to use your SYBR® Green primers for a TaqMan® assay, depending on how they were designed. You would have to design a separate probe to use with your existing primers. Please refer to the guidelines in this manual on “Manually Designing Primers and Probes” for the next steps. If you have Primer Express® Software, you can use that software to design a probe. Please note that restricting the design using the predesigned SYBR® primers may not allow for a successful probe design.
Since SYBR® Green can bind to any double-stranded DNA product, it is important to check your melt curves for the number of peaks. When primers are specific, you should see only one peak. Sometimes you may get extra peaks in a melt curve, which could be due to primer-dimers, a nonspecific product, or gDNA contamination. Please watch this short video for more information on multiple peaks in melt curves and how to deal with them.
If your amplification curves do not have a normal appearance, you may be having an issue with the baseline settings. Please see these examples and suggested solutions in our Real-Time Troubleshooting Tool for more details.
If you are targeting a low-abundance gene, you may have trouble getting Ct values in a good, reliable range (Ct > 32). To increase the sensitivity of the assay, you may want to consider the following:
- Increase the amount of RNA input into your reverse transcription reaction, if possible
- Increase the amount of cDNA in your qPCR reaction (20% by volume max)
- Try a different reverse transcription kit, such as our SuperScript® VILO™ Master Mix, for the highest cDNA yield possible
- Consider trying a one-step or Cells-to-CT™ type workflow (depending on your sample type)
Primer Express® Software
In general, the target Tm of a TaqMan™ probe is 70°C. Using the Primer Express™ Software, you can check the Tm of a MGB or TAMRA™ probe probe.
Within the software, go to Primer Probe Test Tool and then copy/paste in your sequence of interest to see the Tm. If you need to check for a MGB based probe, make sure to choose Document Type: TaqMan™ MGB Quantification. If you need to check for a QSY® or TAMRA™ based probe, make sure to choose Document Type: TaqMan™ Quantification.
Yes, you can use Primer Express™ v3.0.1 on Win7 computers.
You can directly see all the primer and probe sequences from a test design right away using the Primer Express™ Software. Open the software and choose File => New. Choose either TaqMan™ MGB Quantification or TaqMan™ Quantification for Gene Expression Assay design. Copy/paste your gene sequence of interest into the new window and click on the green triangle to get primer/probe designs.
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