Hi,
I extracted RNA today via the trizol method. My 260/280 ratio is 1.95 while 260/230 is 1.44. I washed the pellet using isopropanol and ethanol. The cells grown and RNA extracted from were human cells.
I have somewhat shaky hands especially when anyone watches me extract RNA. However, I did not notice any debris enter my pipette tip during aspiration of the supernatant.
Would it be possible to continue and synthesize cDNA from this RNA for qPCR? Would the qPCR be accurate?
Attached: My Nano Drop
i would suggest before synthesizing cDNA you must give a DNAse treatment to remove any residues of DNA then proceed for the cDNA synthesis and qPCR reaction.
I guess you might have contaminated the samples a bit while extracting with Trizol as EDTA, carbohydrates and phenol have absorbance near 230 nm. Though the 260/230 ratio isn't near 2.0, you could still synthesise cDNA. No worries.
Sounds like some contamination with phenol but since you should do DNase digestion, precipitation, washing, reconstitution and cDNA-Synthesis, your problem is likely to be gone afterwards. Just continue...
As you said your 260/280 is 1.95 it might be because of phenol or other contaminants that absorb strongly at or near 280 nm. For more purity, you can go for washing step and use tris-buffered EDTA solution ( pH of 8.0) instead of nuclease-free water.
Don't worry about shaky hands it will be fine once you become an expert in handling.
Yes, you can proceed for cDNA synthesis and q PCR you will get the quite good result.
Good luck
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