For qPCR experiments I have several target genes and I need to know the amplification efficiencies with the selected primers. So I am taking the risk with perhaps a naïve question.
In order to determine the amplification efficiency of two genes (e.g target and housekeeping gene) is it always necessary to use in vitro transcripts or plasmids of known copy number as RNA (or DNA) standards?
Is the use of increasing dilutions of ng of total RNA from a cell line, which is known to express both genes, also acceptable as reference RNA for amplification efficiency determination?
Laura,
See the recent post at:
https://www.researchgate.net/post/Which_one_should_be_used_to_draw_a_standard_curve_in_qPCR_cDNA_or_PCR_product_of_cDNA?ev=tp_feed_post_xview
It may be of some use to you.
I read also a little bit of the MIQE guidelines and it said that for calibration curves "RNA or DNA from specific biological samples, or internationally recognized biological standards (as they become available) " can be used. However I am confused trying to apply these guidelines to my specific work. So it means that for standard curves preparation I could use as a template, for example cDNA from total RNA isolated of immune-responsive cells treated with LPS, which I previously knew expresses mRNA of a pro-inflammatory cytokine? In such a case, could I also determine the amplification efficiency in order to use the PffafI Method for relative quantification?
I agree there is a lot of confusion on this... but, to me, the best thing to use for standards are the samples themsleves, or template isolated identically to the way the samples were but spiked a known amount of exogenous template.
Another interesting publication concerning this:
http://www.biomedcentral.com/1471-2164/12/118
For relative quantification and the Pfaffl Method, I use a mixture of the experimental samples to generate all target standard curves. Knowing which samples are positive for your targets of interst is always helpful so you don't end up generating very weak standard curves for certain targets.
E.g. IL-8 and TNFa standard curves from a mixture of non-infected/non-'inflamed' samples are often hard to render.
Thanks for your comments and help, I will definitively use the samples for standards preparation.
Dear Laura, just to counterbalance my input/opinion here -- the following publication has ideas you should weigh as well:
http://www.biomedcentral.com/1471-2164/12/118
Dear Jack,
Thanks for the publication, there is truly a whole universe about this topic.
Honestly I am pretty sure about using cDNA experimental samples for target standard curves in my qPCR, first because what I could understood here and https://www.researchgate.net/post/Which_is_the_best_choice_to_determinate_qPCR_amplification_efficiency_cDNA_gDNA_or_plasmid_serial_dilutions?ev=home_person_add_comment_question_target is that the amplification efficiency will not be the same using cDNA biological samples or PCR product standards, especially in the first cycles, which are critical for the further cycles. So if I want to compare the expression levels between cDNA biological samples, why should I use other standards than the cDNA samples itself?
Secondly, it is not the aim of my work to compare the performance of different qPCR plattforms, so it may not be necessary for me to use universal RNA or DNA standards. I am really grateful for your kind attention
Hi Laura,
I agree with you -- using sample-like material (e.g. the samples themselves) seems intuitively the best way to go in [perhaps all] qPCR situations.
When PI's or reviewers believe otherwise, I have published a formula which attempts to reconcile the two universes (when absolutely necessary) .... attached.
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