Is this one-step qRT-PCR?

What do you mean by unstable RT-PCR product? What cT values do you see with the 20uL reaction vs the 10uL reaction? 2.5uL is a large enough volume of input that pipetting error/variation shouldn't cause problems, but 10uL reaction might be below the minimum for your instrument to get an accurate read on the fluorescence. Also, if your sample is very dilute and your cT values are marginal with 20uL reactions, dropping to 10 could put you outside linear dynamic range.

There can be problems with low volumes. Some pcr machines assume a larger volume and when you set a temperature profile the block knows when to stop heating/cooling so that the correct temperature is achieved but at low volumes you may get overshoot of temperatures so your annealing may be low and your denaturation temperature may be high. With very small volumes you can also get evaporation of water into the tube air space so the actual volume of regents is lower than 10ul and all concentrations are changed.Does your pcr machine have a setting for 10ul volumes? If not you may need to make some changes to the pcr profile

Hi Rut Christine Inggriani,

I was told that using a smaller volume can mean that you have more PCR inhibitors present. By using a larger reaction volume, your inhibitors will be more dilute and therefore less likely to interfere with the PCR reaction.

Someone please correct me if I am incorrect. Hope this helps!

Jorji

Hi, I compare ct value between 20uL and 10uL, sometimes I got no amplification in same samples. There's no ct value of IC in my 10uL reaction. On the other hand, ct value using 10uL is slightly lower than 20uL reaction @Laura Leighton

@LauraLeighton

I don't quite follow. 'no ct value of IC'. What does this mean? Is IC your target?

If you have no amplification in some samples, and cT values that are different between 10uL and 20uL reactions despite using proportionally the same amount of input, then the 10uL reactions might be pushing the instrument or assay outside of its capabilities. You might not be able to drop the volume this low and still get acceptable assay performance. What instrument are you using, and are you able to set the reaction volume in the control software? Paul made a very good point about temperature control potentially being incorrect if the instrument assumes a much larger volume of liquid than is actually present.

Hi Laura, I mean I have 4 gene target, Ms2 , S , N, ORF1ab, since I diagnosed covid 19 sample. I using quanstudio 5 from thermo Fisher. Sometimes I got no amplification in my MS2 as IC . Yes you and @Paul Rutland have the point, I didn't change any single method from the protocol using 20 micro, I just change the volume reaction. Do I need to change the method and adjust the temperature if I using 10 micro reaction ? @Laura Leighton

@PaulRutland @Laura Leighton

Yesterday I got very low ct value and my thresshold in Positive control in S gene seems very low (16.64). I usually get ct in 30, Is this also because I using 10 micro reaction? I change my elution RNA volume from 50micro to 30 micro to get more concentrate RNA

A change of ct from 30 to 16 indicates an increase of approx 16000 times the amount of target which cannot be caused by the change of elution volume to 30ul so my feeling is that this is related to the change to 10ul reaction volumes. If all amplifications had changed by a similar amount then better purification of pcr inhibitors might be possible but when the other pcrs are working this seems unlikely