In our recent study, the Ct values of 18S rRNA could vary between 22 to 30 for same FFPE samples in repeated experiments. We have no idea about the underlying major factor that induce this variations since the instruments, reagents, operator are remained the same. Any advice and suggestions will be greatly appreciated!
That's not surprising. FFPE samples are notorious for RNA study because of potential degradation/contamination during fixation process, which is a big cofounding factor for sucessfulRNA experiment. You may need to run a bioanalyzer to check RNA profile of your samples and make sure the RNA sample integrity is ok.
As Tin-Lap Lee said is very important to check the RIN of your RNA sample by bioanalyzer, cause RNA degradation could crucially affect your downstream analysis (gene expression studies for example).
You can also check, if possible, the amplification other reference genes with a supposed stable expression (B-ACT, HPRT, B-2M, etc..), in order to further verify that variation in 18S Ct values is not due to any other factor.
Thank you, Tin-Lap and Alessandro! Based on our experience, working with clinical FFPE samples, the RINs of RNA sample by Bio-analyzer are typically below 6, even sometimes the individual genes' expression level are robust.
- Badges
- Science method