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Questions and Answers
What is the primary role of guanidinium thiocyanate (GITC) in the RNA extraction process?
What is the primary role of guanidinium thiocyanate (GITC) in the RNA extraction process?
After centrifugation in the RNA extraction protocol, where is the total RNA found?
After centrifugation in the RNA extraction protocol, where is the total RNA found?
Which reagent is used to precipitate RNA after the initial extraction with TRIzol?
Which reagent is used to precipitate RNA after the initial extraction with TRIzol?
What is the purpose of washing the RNA with 70% alcohol during the extraction process?
What is the purpose of washing the RNA with 70% alcohol during the extraction process?
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In the protocol, what is the optimal temperature for incubating the pancreatic cancer cells?
In the protocol, what is the optimal temperature for incubating the pancreatic cancer cells?
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Which piece of equipment is essential for assessing the quality of RNA spectrophotometrically?
Which piece of equipment is essential for assessing the quality of RNA spectrophotometrically?
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What should be done immediately after adding TRIzol reagent to the cells?
What should be done immediately after adding TRIzol reagent to the cells?
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What is an important consideration while using the cell culture hood during the experiment?
What is an important consideration while using the cell culture hood during the experiment?
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What is the primary purpose of adding chloroform to the homogenate during the procedure?
What is the primary purpose of adding chloroform to the homogenate during the procedure?
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Why is it necessary to centrifuge the tube at 12,000 x g for 15 minutes at 4°C?
Why is it necessary to centrifuge the tube at 12,000 x g for 15 minutes at 4°C?
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What indicates a successful extraction of RNA after centrifugation and the appearance of the RNA pellet?
What indicates a successful extraction of RNA after centrifugation and the appearance of the RNA pellet?
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What does a 260/280 absorbance ratio of ~1.8 indicate in RNA quantification?
What does a 260/280 absorbance ratio of ~1.8 indicate in RNA quantification?
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What is the significance of the 260/230 absorbance ratio in RNA assessment?
What is the significance of the 260/230 absorbance ratio in RNA assessment?
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What could cause the absorbance at 320 nm to be elevated?
What could cause the absorbance at 320 nm to be elevated?
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How is the dilution factor calculated when diluting a sample with a volume of 20 μl in 1000 μl of dH2O?
How is the dilution factor calculated when diluting a sample with a volume of 20 μl in 1000 μl of dH2O?
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What is an appropriate action to take before measuring the absorbance of the diluted RNA sample at 260 nm?
What is an appropriate action to take before measuring the absorbance of the diluted RNA sample at 260 nm?
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Study Notes
RNA Extraction Procedure
- High-quality RNA is crucial for many molecular techniques, including RT-qPCR
- TRIzol reagent, containing guanidium thiocyanate (GITC), phenol, and chloroform, is used to extract RNA
- GITC denatures DNA, separating it from RNA and proteins
- RNA remains in the upper aqueous phase after centrifugation
- Total RNA is precipitated with isopropanol
- Further purification involves washing with 70% alcohol
Lab Exercise Components
- Students extract RNA from cells using the TRIzol method
- Lab techniques for cell disruption are covered
- RNA quality is assessed spectrophotometrically
RNA Extraction Protocol (Part A)
- Prepare a six-well plate with cells
- Remove media and add 0.5 ml TRIzol reagent to each well for 5-10 minutes
- Homogenize the sample by pipetting
- Add 0.2 ml chloroform and shake vigorously
- Allow the mixture to stand for 2-3 minutes
- Centrifuge at 12,000 x g for 15 minutes at 4°C
- Carefully transfer the aqueous (upper) phase to a new tube
- Add 0.5 ml isopropanol and mix thoroughly
- Incubate the tube at room temperature for 10 minutes
- Centrifuge at 12,000 x g for 10 minutes at 4°C
- Aspirate and discard the supernatant
- Add 500 µl of 70% ethanol and centrifuge at 7500 x g for 5 minutes at 4°C
- Remove the supernatant and air-dry the RNA pellet
- Redissolve the RNA in RNase-free water
- Measure RNA with a spectrophotometer
RNA Quantification and Quality Assessment (Part B)
- Spectrophotometry measures RNA concentration in µg/ml, using the absorbance at 260nm
- Proteins absorb at 280 nm, and nucleic acid / protein ratios are calculated
- A 260/280 ratio of ~1.8 for DNA and ~2 for RNA indicates purity
- Absorbance at 320nm is used to subtract background
- The 260/230 and 260/280 ratio are also calculated
- Dilute the RNA sample and measure its absorbance at 260nm
- Assess absorbance at 320nm to indicate background
- Determine RNA concentration with the formula provided
Protein Contamination
- Assess protein contamination by measuring absorbance at 280 nm
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Description
This quiz assesses your understanding of the RNA extraction procedure using TRIzol. It covers the key steps involved in extracting RNA from cells, including the role of different reagents and techniques for assessing RNA quality. Perfect for students engaging in molecular biology labs.
