Hello fellow researchers,
I'm seeking advice on the potential consequences of deviating from the recommended protocol for RNA stabilization and extraction. Specifically, I collected mouse lungs, chop with scissor and immediately submerged them in RNAlater. However, instead of incubating the samples overnight at 4°C, I directly transferred them to -80°C.
Despite this, my RNA extraction yield and quality appear satisfactory, with Nanodrop (A260/A280 ≈ 2, A260/A230 ≈ 2) and concentrations ranging from 700-1100 ng/μL (following DNase treatment during Extraction).
My question is: Are my downstream qPCR results likely to be affected by this deviation from the standard protocol? Have any of you encountered similar situations, and if so, what were your observations?
Thank you for sharing your expertise and experiences!
Should be just fine. The RNA-later is specifically designed for folks who don't have access to flash freezing in liquid nitrogen or a -80*C freezer.
Thank you for answering.
But I didn't flash freeze just directly stored samples at -80°C.
What about endogenous nucleases. Because RNA later didn't get the time to penetrate inside the tissue.
I got high RNA concentrations but I'm worried what if it contains degraded RNA due to skipping overnight incubation.
Storing samples at -80°C actually helps preserve RNA better, especially since you used RNAlater and DNase I during extraction. This ensures your RNA remains intact and free from contamination.
Beyond this, other factors like proper loading of strips, avoiding contamination, optimizing annealing temperatures, and ensuring primer specificity are equally critical for reliable qPCR results. If your RNA quality are good, your downstream qPCR results are unlikely to be significantly affected by the deviation from the protocol. However, always include proper controls (e.g., no-template controls and positive controls) to validate your results.
@Mohamadhadi Masoumi thanks for your answer.
But I'm just curious if only -80°C actually preserves RNA better then why it's a common practice to SNAP freeze or use RNA later?
Sorry for exaggerating but just curious.
From things that i have experienced, RNA later is mostly used for transporting samples in which RNA is required to be protected or undegraded. Otherwise storing in lab we prefer direct -80 storage
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