I have a question regarding the effect of freeze/thaw of cell pellets on RNA, specifically when trying to amplify low copy targets. I had flash frozen cell pellets to perform RNA extraction at a later point (using Qiagen kit). Due to a freezer issue some of the cell pellets may have briefly thawed and refrozen. After reverse transcription I am using qPCR to detect fairly lowly abundant transcripts. My question is how much degradation could occur from a very brief freeze thaw? If some of the samples thawed, and some did not will this effect the results?
My concern is that since some of the targets are low in abundance, any degradation might affect the amount of RNA of that target, while having relatively little effect on my normalization genes (CD79b or GAPDH) which are in high abundance. Thus, the normalization would not correct for the RNA degradation. Is this a valid concern and can I trust my data? Sadly, this was performed with a double KO mouse that is tough to breed and I do not have more mice right now