Hi All,
Currently, I'm working on a qPCR assay. I am using RT kits for my experiment . the setup that i had before for my assay is this :
For 1 Reaction: Add 2 RT Buffer + 4.5 H20 + 0.5 spike-ins +1 RT-Enzyme For 25 Reaction: Add 50 RT Buffer + 112.5 H20 + 12.5 spike-ins + 25 RT-Enzyme usually i make MM 10% more.
8 µl MM + 2 µl RNA to the reaction mini tube . i have 5 reaction tube so in total = 50 µl reaction. After RT i have 50 µl cDNA.
I could not detect any target in my qpcr, so i turned back to the handbook to re-setup my experiment .
in the handbook they mentioned these points:
- Use a template RNA volume equivalent to 16 µl original serum/plasma for each 20 µl reverse transcription reaction.
- Use a template RNA volume equivalent to 8 µl original serum/plasma for each 10 µl reverse transcription reaction. i have 2 quastions:
1:I do not know what is the best experiment set up according to my previous set up.is amount of cDNA enough?or how i optimize this assay?
2: is the RT calculation correct ?
Any recommendations or solutions would be appreciated.
Thanks
Maria
1 answer
The main thing I would check is whether the RT reaction is actually matching the kit’s recommended input, rather than focusing only on the final 50 µL cDNA volume. The handbook specifies an RNA volume equivalent to 16 µL of original serum/plasma for a 20 µL RT reaction, or 8 µL for a 10 µL reaction. Your current 8 µL master mix + 2 µL RNA setup should therefore be compared carefully with the manufacturer’s intended template input.
I would recalculate the RT components from the kit’s stated final concentrations and confirm that the RT buffer, enzyme, spike-ins, and other components are all at the required levels. Since the primers are already included in the customised plate, it is also worth confirming exactly when and where those primers are introduced.
For troubleshooting, running the recommended RT protocol alongside your previous setup would give you a useful comparison. Include a positive control and RT-negative/no-template controls so you can distinguish an RT problem from issues with RNA quality, inhibition, or low target abundance.
The 50 µL cDNA volume itself is not necessarily insufficient. What matters more is how much original sample is represented in that cDNA and how much cDNA enters each qPCR reaction. For organising multiple conditions and controls, a 96 Well Plate Template can also help document the setup and avoid well-assignment or pipetting errors.
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Are there primers in the RT buffer or is the kit expecting you to use your own primers?
Dear Travis , yes there are primers in the 384 customized plate.so i did not add them.
Regards Maria