RNA transcripts as qPCR standards

[whets66]

Hi,

I have cloned a portion of sequence from my gene of interest in order to make in vitro transcripts for a qRT-PCR assay I am running. I generated the transcripts using MegaScript from Ambion. I purified them using a MegaClear kit from Ambion. I quantified using 260/280 readings on Nanodrop and got about 200-400 ng/ul of RNA on 4 different subspecies. This translated to about 10^11 copies or so. I diluted each to 10^6, 10^5, and 10^4 for starters and didn't see linear progression. The 10^6 duplicates gave ave Ct of 26.7, then 31, then nothing (you'd expect 34.3 here). A similar thing happened to all 3 other transcripts. Some even dropped off completely after 10^6. I have done this work with other RNA viruses and not had this problem. Any advice? Should I add a carrier?

Thanks much.

[Elsie]

Hi,

I have had similar problems recently. Have you figured out any solution to your problem? I have found that standard curves from my transcripts are giving poor slopes and terrible efficiency and I have perfect slope and efficiency etc with other standards on the run so I know it isnt the PCR. I would appreciate any recommendations!!

I have tried larger dilutions, used different pipettes (just in case) and made my dilutions in nuclease free water and in RNA storage solution but to no avail. This is happening with 2 separate constructs.

Thanks!