I did PCR with my cell samples, and it worked very well. Please see 28S of my cell samples in Figure 1. But my tissue samples did not work well, please see 28S of my tissue samples in Figure 2.
My cell and tissue samples were under completely same process. And I checked quality of RNA from both cells and tissues, they were both excellent, the tissue ones were even better. I always stored tissues in 1.5 mL eppendorf tube and put the tubes in liquid nitrogen immediately, then stored them in -80℃ freezer for a longer time. The tissues I used for PCR were just from 10 days ago.
I don't know what was wrong with the PCR of my tissue samples. Would someone please help me out here? Thanks!
Kindly follow the link below for some suggestions
Article DNA Degradation and Its Defects
Could you tell us more about your RNA extraction? Did you use a one-step enzyme, or did you first make cDNA and load that? How were the qualities of the RNA (A260/280 ratios etc). Yields and loads similar? Looks like the bottom set of curves were done using less template.
Kindly follow the link below for some suggestions
Article DNA Degradation and Its Defects
Go for quantification of your samples. Check if there is a difference between the concentration of templates you are using for your reaction. Since you have not mentioned other reaction conditions (starting material, starting concentration, dye/probe, etc), low concentration of template in samples obtained from tissue seems to be the factor responsible for the kind of results you have obtained.
- It is likely in spite of your optimal 260/280 ratios that impurities derived from and replete in your final RNA are inhibiting reverse transcription and thus undermining your PCR
- It is not uncommon for this happen considering that cells are a fairly homogenous prep in comparison to tissues: in particular lipid levels and salt are higher with dirtier tissue preps and salt in particular will inhibit RT and thus delay amplification in real time PCR
- Salt contamination in particular will not be revealed by an ambient 260/280 ratio but will be revealed by a 260/230 ratio of less than 1.0; the ideal is a ratio of >1.5 and ideally nearer 2.0 but anything >1.0 is fit for purpose and anything
- Badges
- Science method