Trizol reagent contains phenol, which may interfere with RNA quantitation if not removed. You may either perform several extractions of your RNA solution with Chloroform or ethanol-precipitate the RNA and wash the RNA pellet with cold 70% ethanol several times.

Hello Tasnim,

The low 260/280 and 260/230 ratios in your RNA samples suggest contamination, likely from proteins and organic solvents, respectively. These issues can arise from incomplete phase separation or insufficient washing during the TRIzol extraction process. To improve the purity of your RNA, you can reprecipitate and wash your samples.

Here are 2 protocols you can use that are available online:

• Ethanol precipitation of RNA/DNA: https://projects.iq.harvard.edu/files/hlalab/files/ethanol-precipitation-of-rna_hla.pdf
• Precipitation of RNA with Ethanol: https://cshprotocols.cshlp.org/content/2020/3/pdb.prot101717.full

Alternatively, you can use a kit for RNA cleanup. Here are some examples:

• RNeasy MinElute Cleanup Kit (Qiagen): https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/rna-clean-up/rneasy-minelute-cleanup-kit
• NucleoSpin RNA Clean-up (Macherey-Nagel): https://www.mn-net.com/fr/nucleospin-rna-clean-up-mini-kit-for-rna-clean-up-and-concentration-740948.50
• RNA Clean & Concentrator (Zymo Research): https://zymoresearch.eu/collections/rna-clean-concentrator-kits-rcc/products/rna-clean-concentrator-5

Adipose tissues have their challenges regarding RNA extraction. You may need to adapt your technique. Here are some articles that discuss this issue:

• Article An Optimized TRIzol-Based Method for Isolating RNA from Adipose Tissue

• Article Optimising total RNA quality and quantity by phenol-chlorofo...

Best of luck,

Sincerely,

YS

Thank you, Victor G Stepanov and Joseph Siblini , for your valuable comments. I will follow up on your suggestions and try to reprecipitate and wash my samples.