Hai Mania,

RNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples can be a challenging process due to the cross-linking and degradation of RNA caused by formalin fixation. However, with the right protocols and reagents, it is possible to extract RNA from FFPE samples. Here's a general procedure for RNA extraction from FFPE tissue samples:

Sample Preparation:

• Obtain FFPE tissue sections (5-10 µm thick) on glass slides. Ensure that the sections contain the desired tissue regions.
• Use a clean scalpel or blade to scrape or cut the tissue sections from the slides into a sterile microcentrifuge tube. Aim for approximately 10-20 mg of tissue.
• If necessary, deparaffinize the tissue by adding xylene to the microcentrifuge tube and incubating at room temperature for 5-10 minutes. Remove the xylene by aspiration or careful decantation.
• Wash the tissue pellet twice with 100% ethanol to remove residual xylene and air-dry the pellet.
• Tissue Digestion:
• Add a suitable lysis buffer (e.g., proteinase K buffer) to the tissue pellet, ensuring complete submersion of the tissue.
• Incubate the tube at an appropriate temperature (e.g., 55-60°C) for several hours or overnight to allow protein digestion and RNA release. Vortex or mix the tube occasionally during the incubation.
• RNA Extraction:
• After tissue digestion, add a phenol-based RNA extraction reagent (e.g., TRIzol) to the tube.
• Mix vigorously to ensure thorough homogenization of the sample.
• Centrifuge the tube at maximum speed to separate the phases: an aqueous phase containing RNA, an organic phase, and an interphase.
• Transfer the aqueous phase (containing RNA) to a new tube, being careful not to carry over any interphase or organic phase.
• Add an equal volume of isopropanol to the aqueous phase and mix gently.
• Incubate the tube at room temperature for a few minutes to allow RNA precipitation.
• Centrifuge the tube at maximum speed to pellet the RNA.
• Remove the supernatant carefully and wash the RNA pellet with 75% ethanol to remove residual salts and contaminants.
• Air-dry the RNA pellet and resuspend it in an appropriate volume of nuclease-free water or RNA storage buffer.
• RNA Quality Control:
• Measure the concentration and assess the quality of the extracted RNA using a spectrophotometer or a fluorometric method.

Hope it will work

Regards

Some of the most popular extraction kits out there: https://www.thermofisher.com/order/catalog/product/K156002 or

https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/rneasy-ffpe-kit

Hello Mania Makhoul

FFPE tissues present many technical challenges in molecular analysis. Formalin fixation leads to crosslinking of nucleic acids to proteins and other cellular constituents, making the extraction of these analytes difficult.

Establishing a reliable and reproducible method of obtaining sufficient amounts of high quality nucleic acids from limited amounts of FFPE tissue remains a major challenge. Currently, several commercial kits are available for the extraction of RNA from FFPE tissue. While the manufacturer’s quality control process ensures a consistent performance under given experimental conditions, each of these kits has distinct performance characteristics in terms of yield and purity.

I would recommend RNeasy FFPE Kit which is specially designed for purifying total RNA from FFPE tissue sections from Qiagen. Please refer to the link below.

https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/rneasy-ffpe-kit

Best

Thank you very much for your answer.

RNA extraction kit from paraffin tissue (FFPE) RNA extraction kit from paraffin tissue (FFPE) is used to extract RNA from all types of human and animal paraffin tissues. The extraction method in this kit is sedimentation. Also, this kit for extracting RNA from paraffin tissue was tested on paraffin tissue samples that were 10 years ago and a very good result was obtained. Products related to RNA extraction kit from paraffin tissue (FFPE) 1. RNA extraction kit from tissue 2. RNA extraction from the plant 3. Mastermix Real Time PCR 4. cDNA synthesis kit 5. RNA extraction kit from blood

See attachment

I did RNA extraction from FFPE samples of breast cancer too. I used the RNeasy FFPE extraction kit, Qiagen. I first got poor yield. However, after adjusting the incubation time, the RNA yield was good. You know it’s a trial and error at first.

Good luck.