The aim of Northern blotting is to evaluate the integrity of target genes in the pharmaceutical industry. I am inquiring about the positive control used to demonstrate the sensitivity of this method, so that, if an aberrant variant is present, it can be concluded to be a true variant above the detection limit. Conversely, if no variant is detected, it can be concluded that no aberrant transcript greater than XX% of the total RNA population.
In the attached, the MCB WCB EOP are all stable cell line (CHO cell) producing rhBMP2. They used rhBMP2 RNA as a positive control at different ratios(1%, 5%, 10% of the total RNA population). For the MCB WCB EOP, the loading amount is not specified. My question is: if the loading amount is 2ug, how much positive control should be loading to achieve 1%, 5%, 10%, and how is this calculated?
Thanks a lot.
A proper positive control for northern blot is which produces strong signals, that indicates the probe is working effectively
Mohammad Iqbal Mir
Thanks for your explaination.
I am wondering how to use the positive control to assess the limt of detection as attached. and how to calculate the loading amount of positive control to achieve such like 5%of total RNA population. I this papar they conclude that a single rhBMP-2 transcript of the expected size with no evidence of aberrant transcript greater than 1% of the total RNA population.
looking forword for you insight~~
I would think the original paper would tell you how they did the calculations. But my guess is that this is merely a dilution series, they took their 2ug of the known positive control sample and then made a 10-fold, 20-fold and 100-fold dilution of that sample and loaded it on the gel.
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