Hello, Im have a time working with qPCR and i have a lot of variability between tecnical replicates and biologicals ones. I made a lot of changes on my protocols and my results going better but not enough. I make a mix with sybr green, primers and cDNA, and from this mix i take for two tecnical replicates (one mix on two wells) (i made all of this taking care of not pippeting less than 5 uL as recommended) , so my tecnical replicates are identical, at way I'd wait no deferences in Ct values, but i have differences at point one of the well amplificate and another one doesnt. This tend to happen with the GOIs wich are less abundant, but not currently with HKPs. i dont know its maybe a problem with the qPCR equipment bc i think it not explainable to the point of pippeting. what do u think?
Dear Diego
As you explained you may not have a problem with pippeting. According to my experience lower level of transcription possibly affect the repeatability. Also the reaction condition (e.g. sub-optimum annealing temperature) can also affect the amplification efficiency which as a result may affect the repeatability. Therefore, I would recommend:
1-Try to optimize your reaction with your GOIs
2-If possible, make new cDNA by incorporating more RNA input
These will improve your reaction kinetics and also repeatability.
Good luck
Hey Diego Zavala ,
did you always use the same well-positions for your qpcr and is always the same position "curios"? I am asking this, because we often had problems with not working positions in the cyclers anymore. Because of not heating anymore we did not get any result. This could be an easy explanation to your question. Probably aks your colleagues if they have the same problems.
If its not a technical problem, try the suggestions of Ali.
Furthermore you could also try new primers to get a better working qPCR or if you did not try it already, do a gradient PCR to determine the best conditions for your amplicon.
Hope this helps a bit.
Greetings,
André
If you have very low copy number genes of interest, then you may need to increase the amount of your cDNA in each reaction.
Check that you are not seeing evaporation from any of the samples that are not showing any amplification. Evaporation and/or air bubbles will give you poor results.
I've also had issues with certain wells not working well (problem with the temperature block in the thermocycler).
Good luck!
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