MD105 Lab 2: RNA Extraction Protocol PDF

Kyriakou TC. / Markantoni D./Andreou M. F2024 MD105 - Cellular and Molecular Biology Lab Exercise #2: RNA Extraction: Quantification of RNA and sample quality assessment Introduction Obtaining high-quality RNA is the first, and often the most critical, step in performing many molecul...

Kyriakou TC. / Markantoni D./Andreou M. F2024 MD105 - Cellular and Molecular Biology Lab Exercise #2: RNA Extraction: Quantification of RNA and sample quality assessment Introduction Obtaining high-quality RNA is the first, and often the most critical, step in performing many molecular techniques such as reverse transcription real-time PCR (RT-qPCR). (1) The basic principle of the method is the isolation and separation of RNA from DNA and protein after extraction with a solution called TRIzol reagent. TRIzol is an acidic solution containing guanidinium thiocyanate (GITC), phenol and chloroform. GITC irreversibly denatures the DNA which is a process in which proteins or nucleic acids lose their quaternary, tertiary and secondary structure, which are present in their native states. This is followed by centrifugation. (2) Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. (3) Total RNA is then recovered by precipitation with isopropanol. (4)Finally, RNA washing and purification is done using 70 % alcohol. Students will: Extract cellular RNA using TRIzol reagent method Identify the role of specific reagents and equipment in the extraction of RNA Understand cell disruption methods and practice basic lab techniques To assess the quality of RNA spectrophotometrically 1  Kyriakou TC. / Markantoni D./Andreou M. F2024 PROTOCOL Material/Equipment: 1. Pancreatic cancer cells (MIA PaCa-2 cancer cell line) 2. Cell culture hood 3. Cell Incubator Optimal temperature (37.0°C), humidity (>90%) and C02 (5%) 4. Cell culture vessels (6-well plates) 5. Serological pipettor and pipettes of various capacities 6. 70% ethanol 7. Waste container 8. Centrifuge machine 9. TRIzol Reagent 10. Chloroform 11. Isopropanol 12. RNase-free water 13. Spectrophotometer 14. Cuvettes EXPERIMENTAL PROCEDURE PART A – RNA Extraction 1. Take a six-well plate stored at the incubator. (Make sure the incubator is closed properly). 2. Remove media from a 6-well plate (from your wells) and add 0.5ml of TRIzol Reagent to each well for 5-10mins. 3. Pipette the homogenate up and down and place it in a tube. 4. Leave the tube containing the homogenate on the bench at room temperature for 2 minutes. 5. Add 0.2 ml chloroform. Securely cap the tube containing the homogenate and shake it vigorously for 15 seconds. 6. Leave the tube containing the homogenate on the bench at room temperature for 2–3 minutes. 7. Centrifuge at 12,000 x g for 15 minutes at 4°C. What happens to the sample? 8. Carefully transfer the upper, aqueous phase to a new tube. WHY? 9. Add 0.5 ml isopropanol and mix thoroughly by vortexing. 10. Place the tube on the bench at room temperature for 10 minutes. 11. Centrifuge at 12,000 x g for 10 minutes at 4°C. 2  Kyriakou TC. / Markantoni D./Andreou M. F2024 12. Carefully aspirate and discard the supernatant. The RNA pellet is often visible as a gel-like or white pellet at the bottom of the tube. 13. Add at least 500μl of 70% ethanol and centrifuge at 7500 x g for 5 min at 4°C. 14. Remove the supernatant completely, and briefly air-dry the RNA pellet. 15. Redissolve the RNA in an appropriate volume of RNase-free water. 16. Measure your sample at the spectrophotometer. Part Β: Quantification of RNA and qualitative assessment Spectrophotometer gives out the concentration of the sample (DNA, RNA, or protein in μg/ml). DNA and RNA absorb at 260 nm. Proteins absorb at 280 nm of wavelength. From the difference in intensity (I/Io) A is calculated. By applying Beer’s Lambert’s equation, with set length (1cm) and known extinction coefficient for RNA 0.025, the concentration is evaluated. 260/280 and 260/230 absorbance ratios correspond to the ratio of nucleic acids/proteins and nucleic acids/salts in the solution respectively. It is an indication of purity. 260/280 should be ~1.8 for DNA and ~2 for RNA. Anything less than 1.70 should be avoided. 260/230 are commonly around 2.0 – 2.2. Absorbance at 320 is an indication of dirty cuvette or random debris introduced during the extraction and thus it is subtracted as the background from other wavelength readings. Sample Reading 1. Dilute RNA sample with unknown concentration: 20/1000 (final volume 2500μl) with dH2O. What is the dilution factor? 2. Mix well and transfer dilution in a separate cuvette. 3. In a new cuvette, transfer 2500μl of dH2O which will be used as your blank (sample without RNA). 4. Adjust the spectrophotometer at 260nm and blank it. 5. Measure the absorbance of the diluted RNA sample at 260nm. Write down the absorbance value for dilution. 6. To take into consideration any impurities also read the absorbance at 320nm. 7. Adjust the spectrophotometer at 320nm and blank it. 3  Kyriakou TC. / Markantoni D./Andreou M. F2024 8. Measure the absorbance of the dilution of the RNA at 320nm. Write down the absorbance values for the dilution. Protein contamination assessment: 9. Adjust the spectrophotometer at 280nm and blank it. 10. Measure the absorbance of the diluted RNA at 280nm. Write down the absorbance values for each dilution. Determination of RNA concentration 1. RNA concentration calculation (impurities considered): RNA concentration (μg/ml) = [(A260 – Α320)/0.025] x Dilution factor Total RNA amount (μg) = RNA concentration (μg/ml) x final sample volume (ml) 2. Quality assessment of the RNA sample: RNA purity (Α260/Α280) = (A260 – Α320) ÷ (Α280 - Α320) 4