Recently, I have been investigating of expression of Sall-4 in two different types cancers. Due to of acceptable melting peak of Beta Actin(and Cq=17), I am sure the quality of cDNA is pretty good.
#PCR #QPCR
To compare transcript levels of genes expressed at a very low level, one can also enrich the targets by including a pre-amplification step.
I have included one reference here. There are many others as well:
https://www.takarabio.com/learning-centers/real-time-pcr/overview/qrt-pcr-to-detect-rna-present-at-low-levels
To compare transcript levels of genes expressed at a very low level, one can also enrich the targets by including a pre-amplification step.
I have included one reference here. There are many others as well:
https://www.takarabio.com/learning-centers/real-time-pcr/overview/qrt-pcr-to-detect-rna-present-at-low-levels
If possible try to change the primer set and check the Rt-pcr with these primers only, without adding cDNA and check CT values whether it is giving low ct value because of primer dimers or not.
Pre-amplify the targets. Optimize the reaction (using different Taq polymerase preparations and different sets of primers). Perform the RT-PCR / RT-qPCR. A quick way is to check for primer-dimers is to perform gel electrophoresis for the amplified products to visualize the bands. If primer-dimers are present, they will usually appear as a band below the 50 base pair regions.
Redesign your primers:
https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome
Use publicly available online tools to predict primer dimerization: ie http://www.premierbiosoft.com/qOligo/Oligo.jsp?PID=1.
(or similar tools)
Just paste the primer sequences and analyze.
In the resulting result table, the delta G of cross dimers, self dimers and Hairpin should ideally be 0.0, or at least not more negative than -1.0.
In subsequent tm analysis, the primer dimer peak should be gone
First thing you may try is to start with higher concentration of template RNA (1-2 ug of total RNA) for cDNA synthesis (please make sure you use appropriate kit for this quantity of template). As per my understanding, you should get Ct value of ~30 even if you start with single template copy and using SYBR, if primers are specific, you could see dimer only >35 cycles.
Alternatively, you may consider using more sensitive TaqMan / Molecular Beacon methods.
Fisrtly, you must avoid primer dimers in yor qPCR. I recommend to redesign the primers using a tool like Beacon Designer from Premier Biosoft (there is a free online tool here http://www.premierbiosoft.com/qOligo/Oligo.jsp?PID=1).
If your target is expressed at a very low level you can try to make cDNA synthesis with a high-efficiency reverse transcriptase, like the Superscript IV (invitrogen).
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