I am using a ChIP protocol that uses an ON reverse cross-linking step at 65C. This is common is many protocols, as it is suppose to clear the chromatin of proteins bound to it.
However, when I later test expression of the same sample that is either reverse-cross linked ON or not reverse-cross linked ON. The Cq values / expression of my target is exactly the same. I was wondering if anyone has any thoughts on this, because it worries be a little bit. Explanations I thought of:
1. My cross linking step (10 min at RT, 1% formaldehyde) is not cross linking properly. Therefore, the ON reverse-cross linking does not make a difference, as the sample is not cross-linked in the first place.
2. The PCR is able to access the parts of the chromatin with the proteins bound to it, so a reverse-cross linking step is redundant.
3. Proteinase K treatment (10 min at 56C) is performed after reverse-cross linking and before DNA isolation and is enough to remove all proteins.
I was wondering if anyone has any thoughts on this, because it worries be a little bit.
Hoping for a good discussion,
Best,
Bart
For the full story: Reverse cross linking is done in elution buffer. I use columns for DNA isolation, but the elution buffer interferes with the column extraction (maybe SDS or pH?). Therefore, I first precipitate the DNA, dilute in PBS and continue with column extraction.
I guess proteinase K treatment and additional DNA precipitation/column extraction is sufficient to remove the proteins. In all protocols I used, the 65°C de-crosslinking step was only performed for 2h and proteinase K was added. Do you just cook the sample in elution buffer without adding anything?
I apologize for not clearly understanding your specific question. However, to answer your title, if RT-PCR is your objective, my simplest answer is "no".
- Badges
- Science method