hello everyone, i hope you can help me about this troublesome quest:
-I will study 2 conditions with 3 biological replicates (3 samples in control vs 3 samples in treatment) (6 samples in total)
-3 reference genes, and 8 problem genes.
-3 technical replicates for each gene per sample.
this obviously means I wont be able to put all the arrangement in a 96 well qPCR plate.
anyone could refere me to good literature about what considerations should be taken before start?
I have heard about inter run calibrators, other mention use reference genes in both plates, but this is out of possibility since 3 ref genes take 70% of my first plate.
I hope anyone can give me some oppinion and references about this matter.
thank you very much
Hi Gregory,
I don't know if you have been able to find anything online. Where I first started doing qPCR I read the manuals that came with the qPCR machine as they normally give you examples of the types of experiments you can do.
I'm often looking at mulitple biological samples and multiple genes of interest and generally use relative quantification method to save having to use wells to do a std curve. From my understanding, on a 96 well plate, if you do your samples in triplicate, then 18 wells will cover you for 1 primer set/gene of interest. You should, therefore, be able to look at 3 reference genes and 2 problem genes per plate (and still have 6 wells for your water blank CT and -RT control from the cDNA synthesis reaction). This means that you will require 4 96-well plates to investigate your 8 problem genes and be able to normalise these against your reference genes.
Alternatively, if you have the right heat block, you could use 384-well plates, which will allow you to test more 'problem genes' per plate.
Here is a link from 1 manual below - p11 give the example of different tissues on 3 different plates; however, this could equally be the same tissues, but different genes being investigated:
https://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042115.pdf
Hope this helps.
Good luck!
hello, Chinmaya, thank you for your answer,
what does it mean to make new cDNA? normalizing every batch? even if it comes from the same RNA extraction?
in my protocol I perform retrotranscription in 25 uL batches, which means I just have enough cDNA to perform PCR for only 4 genes per retrotranscription batch.
Does this mean I have to perform another normalization for the next batch of cDNA and so on?
hello Chinmaya,
I really appreciate your answers and help, I still have a doubt in mind:
How well supported is this method? I have seen publications doing it, would It be a problem for referees to use this method of dilutions? or is it a normalized practice?
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