MD105 Cellular Biology Lab Exercise 2 RNA Extraction 2024 PDF

MD105 - Cellular Biology Laboratory Lab Exercise 2: RNA Extraction: Quantification of RNA and sample quality assessment Fall Semester 2024  Objectives Theoretical Background: Introduction to RNA Extraction Cell lysis and purification Material...

MD105 - Cellular Biology Laboratory Lab Exercise 2: RNA Extraction: Quantification of RNA and sample quality assessment Fall Semester 2024  Objectives Theoretical Background: Introduction to RNA Extraction Cell lysis and purification Materials and Equipment Methodology and Protocol: Isolation and separation of RNA using TRIzol Precipitation of RNA using isopropanol Purification of RNA using ethanol Spectrophotometry Principles – Sample quality assessment  Introduction to RNA Extraction Ribonucleic acid (RNA) is one of the three major biological macromolecules that are essential for all known forms of life (along with DNA and proteins). RNA Extraction is the purification of RNA from biological samples. It is often difficult to isolate intact RNA as RNases, a group of enzymes that degrade RNA molecules are abundant in the environment and it is difficult to remove or destroy them completely. RNA isolation requires cautious handling of samples, good aseptic technique, and RNase free solutions during the extraction. Extracting high quality and quantity of RNA from cells in monolayer is essential for many gene expression experiments. Introduction to RNA Extraction  Cell Lysis: Reagent-based Cell Lysis methods: Reagent-based and physical methods. In physical methods, cell membranes are physically broken down by using shear or external forces. In reagent-based methods, specific formulated lysis buffers are used to disrupt the cell membrane. Today we will use TRIzol Reagent: It is a mono-phasic solution of phenol and guanidine isothiocyanate. It maintains RNA integrity during tissue homogenization, while at the same time disrupts and breaks down the cells and cell components. It is used for adherent cell detachment, and it is commercially available.  Materials and Equipment 1. Cell culture hood 2. Cell Incubator [optimal temperature (37.0°C), humidity (>90%) and C02 (5%)] 3. Cell culture vessels (6-well plates) 4. Serological pipettor and pipettes of various capacities 5. 70% ethanol 6. Waste container 7. Centrifuge machine (set at 4°C) 8. Vortex 9. TRIzol Reagent 10. Chloroform 11. Isopropanol 12. RNase-free water 13. Spectrophotometer and cuvettes or NanoDrop Pancreatic cancer cells Are you ready to begin?  Protocol 1. Remove media from a 6-well plate (from your wells) and add 0.5ml of TRIzol Reagent in each well. Leave the sample at the bench for 10 mins. 2. Pipette the homogenate up and down and place it in a tube (all in one). Leave the tube containing the homogenate on the bench at room temperature for 5 mins. 3. Add 0.2 ml chloroform. Securely cap the tube containing the homogenate and shake it vigorously using vortex for 15 seconds. 4. Leave the tube containing the homogenate on the bench at room temperature for 2–3 minutes. 5. Centrifuge at 12,000 x g for 15 minutes at 4°C. What happens to the sample?  Centrifugation Please remember ! 1. If it’s not balanced it will be unstable, and it will not spin at the right speed. 2. It causes denser particles to settle to the bottom of the tube, while low-density substances rise to the top !  Phase Separation Aqueous phase: RNA Interphase: DNA Organic phase: Protein and lipids Carefully transfer the upper, aqueous phase to a new tube.  Protocol 7. Carefully transfer the upper, aqueous phase to a new tube. WHY? 8. Add 0.5 ml isopropanol and mix thoroughly by vortexing. 9. Place the tube on the bench at room temperature for 10 minutes. 10. Centrifuge at 12,000 x g for 10 minutes at 4°C. 11. 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. 11. Add at least 0.5ml of 70% ethanol, break the pellet, and centrifuge at 7500 x g for 5 min at 4°C. 12. Remove the supernatant completely, and briefly air-dry the RNA pellet. 13. Redissolve the RNA in an appropriate volume of RNase-free water (30μl). 14. Measure your sample at nanodrop spectrophotometer.  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.  Sample Reading 7. Adjust the spectrophotometer at 320nm and blank it. 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. Salt contamination assessment: 11. Adjust the spectrophotometer at 230nm and blank it. 12. Measure the absorbance of the diluted RNA at 280nm. Write down the absorbance values for each dilution.  Spectrophotometer Beer-Lambert Law (Principle-Instrumentation-applications) The relationship between I and Io (i.e., the Absorbance) depends on the path length (L) and the concentration of the molecules in the solution (C). A= ε L C C= A/ε L = A/0.025 A= absorbance (calculated by the A=log (I/Io) ε = absorptivity or extinction coefficient (a constant, ability of specific molecules to absorb light) L= length of path (the light pathlength through the cuvette is 1cm) C= concentration Sample Quality Assessment  Concentration and purity assessment ▪ 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 (10mm), 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 Quality Assessment What is measured? 1. Concentration => ng/μl 2. Purity => Protein (260/280) and Salt (260/230) Optimal ratios: Sample 260/230 260/280 RNA 2.00 (2.00-2.20) 2.00 (1.80-2.00)  Questions! What are the four basic steps of the RNA Extraction process? Please explain briefly. How do you determine the concentration of isolated human RNA? Can you list any problems that you could have in the lab performing this experiment? Can you think of any application of this procedure in the medical field?  Determination of RNA concentration 1. RNA concentration calculation (impurities considered): RNA concentration (μg/ml) = [(A260 – Α320)/0.025] x DF Total RNA amount (μg) = RNA concentration (μg/ml) x final sample volume (ml) 2. Quality assessment of the RNA sample: Α260/Α280 = (A260 – Α320) ÷ (Α280 - Α320) Α260/Α230 = (A260 – Α320) ÷ (Α230 - Α320)