I will be using the primers for gene expression using RNA samples, but can I validate them first using gDNA or does it have to be with RNA, or both?
Hi!
To validate primers for qRT-PCR, we usually use three controls:
1) Positive (e.g. control RNA from Agilent) is to validate that primers are annealing to RNA.
2) Negative (ddH2O) is to exclude contamination and to check can primers form any products without template.
3) gDNA is to exclude primer binding with DNA. However, if you design primers within exons, they will bind with DNA as compared to primers designed to span an exon-exon junction.
Thus, you can validate primers using gDNA, if they have been designed on exon-exon junction.
Use of a single pool of all cDNA samples to generate a standard curve seems to fairly common for calculation of reaction efficiency. ABI instruction manual (see attached, p.11), however, does not have any special recommendations regarding the nature of the template, as long as it is considered to be inhibitor-free and sufficiently concentrated. So if you don't have a lot of cDNA or your transcript is in a low copy number that is not enough to prepare serial dilutions of the sample, gDNA should work fine.
A more accurate quantification might be obtained by performing assay validation on dilutions of the individual samples. The rationale for this approach is that experimental-condition or sample-specific differences in PCR efficiency may exist and be attributable to differential copy-number changes, sample-specific expression of antisense, homologous or fusion transcripts, or the differential presence of other inhibitors.
Thank you all for the suggestions and help. Olga, thanks for the attachment. It is very informative.
I have tried using cDNA dilutions of my actual samples but the copy number is too low to get more than the first dilution.
Also, I need to assess my genes for samples collected at different time points for treated and untreated samples. Do I have to validate the primers every time I look at a new time point or would it suffice just doing one?
Validating primers once would suffice. It would be better if you can prepare the "stock 1" by pooling small amounts of cDNA from all of your samples.
Dear Marte,
If your application is to check the gene expression in eukaryotic system, then your primers must have been designed covering two exons excluding the intronic region. In such case, using gDNA will not be helpful, however, you can use gDNA when you intend to check gene expression in prokaryotic system.
In our laboratory, as soon as we design using softwares, or obtain the primer sequence from the Qprimerdepot or from the reported articles, we check for the specificity using MFE Primer-2.0 (biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0/) against organism of interest, and check for its possible amplicons for both gDNA and mRNA. If it gives amplicons for gDNA we will ignore the primer pairs and will look for another one.
Also, as soon as we receive the primers, we used to run around 5 ul of stock primers in 2% Agarose gel and check for the intensity using ImageJ. If the intensity varies, we used to adjust the dilution of intermediate stock to have equal intensity in both forward and reverse primers.
These steps will give you proper selection of primers and subsequent success in your experiments in all kinds of PCR applications. This will be more helpful while validating the PCR efficiency in gene expression analysis.
Also keep positive and negative controls as suggested by Mr. Denisov, and you can also have pooled cDNA samples from different tissue types (if possible) to validate your primers, since gene expression is most of the time exhibits spatial and temporal specificity.
All the best wishes for your qPCR primer validation experiments.
Regards,
Rajkumar
Speaking of pooling my samples, is it recommended to pool my different RNA and then converting this single tube to cDNA, or converting the individual samples to cDNA first and then pooling? Does it matter?
I apologize if these questions seem very elementary. I appreciate everyone's time to answer my questions. I am learning a lot with each of your responses.
It would be better to convert RNA of different samples to cDNA and then to pool it with equal proportion.
Also, correct me if I'm wrong, but I would only be pooling samples from my untreated group (control) to validate the primers, correct? If I'm comparing treated versus untreated groups for gene expression, there might be a case where this gene may not be detectable if the treatment worked, hence can't use treated samples to check for my primers.
If you are sure that your treatment is gonna affect the expression of your gene negatively, then you can use only the pool of control RNA. Else, if you are not sure about the outcome, then I would suggest you can pool both control and treated group in an equal proportion.
If you have taken 1ug of RNA for cDNA conversion in each group, then you can have equal volume, if the initial concentration varies, then you need to calculate and have equal concentration.
equal volumes of cDNA, but make sure that when you convert RNA to cDNA, you use the same input amount (say, 1ug RNA) in all your samples. This insures that the RT reaction efficiency does not vary among your samples, since differences in template concentration and co-purified inhibitors may differently affect the dynamics of RT.
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