I am testing a Maxima First Strand cDNA Synthesis (Thermo-Fermentas). It seems fine except that 1/10 or 1/20 dilutions of cDNA for Q-PCR show lower CTs than expected; it is like the PCR is enhanced. Although, it sounds good that is not right as it could over/underestimate Q-PCR quantification. To avoid this problem I am diluting cDNA at least 1/50.
Example: (fake numbers but this happens with all pairs of primers)
Dilution 1/10 CT 16
Dilution 1/40 CT 21
Dilution 1/160 CT 23
Dilution 1/640 CT 25
Dilution 1/256 CT 27
Does anyone have an explanation?
Dear Pedro, as I can understand you are experiencing unwanted CTs i.e. improper Standard Curve. We faced problems with lower dilutions i.e. concentrated samples. The quick answer was the use of fairly diluted samples.
Agreed with @Harsh Panwar, Try to do serial dilutions, and in repeats.
When doing the serial dilutions, mix properly after adding the template from the prior solution.
And make sure the pipette is adjusted to the proper measurement each time - this is the reason I dislike Gibson pipettes. The volume adjustment is linked to the push button, and after a few consecutive pushes, the volume can deviate slightly.
I agree with @Harsh Panwar and @Jun Hoe Lee:
Sometimes when you work with very low concentrations of cDNA (in your serial dilution) there can be some pippeting problems that can increase your error and give you an efficiency greater or lower than 100%. It's not the same pippeting 100 more copies of cDNA in a mixture that has 1 million of copies of cDNA (that would be a 0.01% of error) that pippeting 1 more copy of cDNA in a mixture of 100 copies of cDNA (that would be a 1% of error). Therefore, in this type of experiments if you work with a very low concentration of cDNA, the efficiency can be usually different to 100%. So avoid too big dilutions.
The is no mixing, pipeting, tecnical problem, or problems with low dilutions...is concentrated cDNA which amplify too much!
As Harsh Panwar said, the thing is that if you take data from all dilutions for a standar curve or efficiency calculation - it does not work, If you remove concentrated cDNA is perfect.
Is like something is carried from "cDNA reaction mixture" and the q-PCR is enhanced.
I do not know if anyone obserevd this issue before, or if there is another logical expalnation.
Anay case, thanks for answering.
Hola Pedro,
Si he entendido bien, tus valores de DeltaCt en diluciones seriadas son menores de los esperados (aunque no interpreto lo mismo con los datos de Cts que muestras). En este caso existen diversos escenarios, quizas los mas plausibles son la existencia de DNA contaminante o la inespecificidad de los oligonucleotidos utilizados. Esto puede generar eficiencias de amplificacion mayores a las teoricas (>105-110%) aceptables para una cuantificacion con correccion de eficiencia. Te recomiendo la utilizacion de diferentes controles para evaluar esta posibilidad.
Si los valores de Cts que comentas en el ejemplo son aproximados, parecen indicar que tienes un transcrito altamente representado en tu muestra de cDNA. Para estos casos, la mejor opción es usar una dilución de tu reaccion de RT como working solution.
Si quieres que comentemos este efecto en mayor profundidad, puedes ponerte en contacto conmigo.
Saludos!.
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