Hi everybody,
I have been doing RNA isolation from human serum, but when measuring the isolate with NanoQuant equipment, the ratio obtained is very low. How the improve the Ratio of RNA isolated from human serum?
I appreciate if you can help me!
Paul Rutland
Usually the 260/230 ratio is low because the absorbance of guanidine salts is high reducing the ratio. Try using less serum and also include an additional wash step after the rna binding step to remove excess salts
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Alireza Mohebbi
Use fresh serum: RNA in serum is relatively unstable and degrades quickly over time, so it is important to use fresh serum for isolation. Optimize the isolation protocol: There are many different RNA isolation protocols available, and it is important to optimize the protocol to ensure maximum RNA yield and quality. Some popular RNA isolation methods for serum include using commercial kits (such as the Qiagen miRNeasy Serum/Plasma Kit) or the TRIzol reagent. Use RNase inhibitors: RNases are ubiquitous and can easily contaminate RNA samples, so it is important to use RNase inhibitors during the isolation process. This can help prevent RNA degradation and improve the yield and quality of the isolated RNA. Ensure proper storage: RNA is sensitive to temperature, pH, and other environmental factors, so it is important to store the isolated RNA properly to prevent degradation. RNA should be stored at -80°C or in liquid nitrogen for long-term storage, and at -20°C for short-term storage. Measure RNA concentration and purity: Once RNA is isolated, it is important to measure its concentration and purity using a spectrophotometer or a specialized RNA quantification tool such as the Qubit Fluorometer. A low RNA concentration or poor purity can indicate RNA degradation or contamination and may require repeating the RNA isolation process.
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Muhannad Alruwaili
I agree with Paul and Alireza. If the above tips are not working, You can try sodium acetate RNA precipitation, as it helps increase the ratio. You have to remember that sometimes if RNA in a sample is low, that will result in a low ratio.
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