Hi all,
I am planning to do a RT-PCR followed by qPCR starting from 100 ng of total RNA. The way we do it in our lab is we dilute the 20 uL of RT reaction containing 1 ug of total RNA 1:4 and use 2 uL of it per 12.5 uL of reaction per well for a 96-well plate for qPCR. Will this protocol work for 100 ng of starting RNA conc.? Will it be too low to detect? We use myScript Sybr Green kits from Bio-Rad. Also, I am starting with low amount of total RNA conc, (in the range of 6-30 ng/uL).
Please tell me your opinions! Thank you!
The minimum amount I used to run RTqPCR is 1ul, you need to make sure the pipette is accurate!
The minimum amount I used to run RTqPCR is 1ul, you need to make sure the pipette is accurate!
I have used Sybr Green a lot. The amount you have should be enough. If you don't have a lot of experience pipetting a good tip can be to dilute it in 40uL of water instead of 20uL and use the double of volume of the dilution for the reaction (double diluted volume=same amount of initial cDNA). The differences between repetitions are smaller when you don't have a lot of experience if you pipet larger volumes. Good luck!
Thank you for your answers guys! I was actually referring to the minimum amount of cDNA (ug) or range of it and not the volume! Do you have any suggestions on that?
I had to stick to low concentration as my RNA was isolated from Laser Captured Microdissection, I have done nearly thousand Q-PCR reactions with as little as 3ng/reaction and it worked absolutely fine.
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