MD105 - Cellular Biology Lab: RNA Extraction

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Questions and Answers

What is the primary purpose of using TRIzol Reagent during RNA extraction?

To maintain RNA integrity while breaking down cells (correct)

To increase the temperature of the cell culture

To precipitate RNA from the solution

To enhance RNA synthesis during cell lysis

Which of the following best describes RNases?

Enzymes that enhance RNA stability

Chemical agents that assist in RNA extraction

Enzymes that degrade RNA molecules (correct)

Proteins that inhibit RNA purification

Which method is NOT a type of cell lysis described in the context of RNA extraction?

Physical methods

Chemical methods

Reagent-based methods

Thermal methods (correct)

What role does the spectrophotometer play in RNA extraction?

It determines the concentration and purity of RNA. Signup and view all the answers

When extracting RNA using TRIzol, how long should the sample be left at the bench after adding the reagent?

10 minutes Signup and view all the answers

Which of the following is essential for maintaining RNA integrity during extraction?

Employing RNase-free solutions Signup and view all the answers

Which component is NOT included in the list of materials needed for RNA extraction?

Acetone Signup and view all the answers

What is the function of isopropanol in the RNA extraction process?

To precipitate extracted RNA Signup and view all the answers

What is the purpose of adding chloroform during the sample preparation?

To facilitate phase separation of the sample Signup and view all the answers

Which component of the sample is most likely found in the aqueous phase after centrifugation?

RNA Signup and view all the answers

Why is it important to balance the centrifuge before running the sample?

To prevent damage to the centrifuge Signup and view all the answers

What happens to the RNA pellet during the ethanol washing step?

It becomes larger and more visible Signup and view all the answers

After measuring the absorbance at 260nm, why is it necessary to also read absorbance at 320nm?

To assess potential protein contamination Signup and view all the answers

How is the dilution factor calculated when diluting an RNA sample?

Final volume of solution / Initial volume of RNA sample Signup and view all the answers

What is the role of isopropanol in the RNA extraction process?

To precipitate RNA from the solution Signup and view all the answers

What indicates that the RNA pellet is present at the bottom of the centrifuge tube?

A gel-like or white pellet appears Signup and view all the answers

What is the purpose of measuring absorbance at 280 nm in RNA analysis?

To assess protein contamination Signup and view all the answers

According to Beer-Lambert Law, what is required to determine the concentration (C) of a solution?

Absorbance values and the extinction coefficient Signup and view all the answers

What does a 260/280 absorbance ratio of less than 1.70 indicate?

Possible protein contamination Signup and view all the answers

Which of the following is true regarding the optimal 260/230 and 260/280 ratios for RNA?

260/230 ratio should be between 2.00 and 2.20 Signup and view all the answers

What is indicated by a high absorbance measurement at 320 nm?

Dirty cuvette or debris Signup and view all the answers

What is the derived formula for concentration using absorbance according to the provided information?

C = A / (ε * L) Signup and view all the answers

Which wavelength is primarily used to assess the purity of RNA against salt contamination?

230 nm Signup and view all the answers

What is the typical absorbance ratio at 260/280 for good quality RNA?

2.0 Signup and view all the answers

Study Notes

MD105 - Cellular Biology Laboratory - Lab Exercise 2: RNA Extraction

Course: MD105 Cellular Biology
Lab Exercise: RNA Extraction: Quantification of RNA and sample quality assessment
Semester: Fall 2024
Objectives:
- Understand the theoretical background on RNA extraction. This involves the introduction to RNA Extraction, cell lysis and purification, materials, and equipment required.
- Learn the methodology and protocol. This includes RNA isolation techniques using TRIzol, precipitation of RNA using isopropanol, and purification of RNA with ethanol
- Understand spectrophotometry principles and sample quality assessment
Introduction to RNA Extraction:
- RNA is one of three major biological macromolecules crucial for life, along with DNA and proteins.
- RNA extraction involves purifying RNA from biological samples.
- Isolating intact RNA can be challenging due to the abundance of RNases (enzymes that degrade RNA) in the environment, and their difficult removal.
- RNA extraction requires careful sample handling, good aseptic technique, and RNase-free solutions.
- High-quality, high-quantity RNA extraction from monolayer cells is critical for gene expression experiments.
Cell Lysis: Reagent-Based:
- Cell lysis methods include reagent-based and physical methods.
- Physical methods use shear or external forces to break cell membranes.
- Reagent-based methods employ specific lysis buffers to disrupt cell membranes.
- TRIzol reagent is a mono-phasic solution of phenol and guanidine isothiocyanate, which preserves RNA integrity while breaking down cells.
- TRIzol reagent is commercially available and useful for detaching adherent cells.
Materials and Equipment:
- Cell culture hood
- Cell incubator (37.0°C, >90% humidity, 5% CO2)
- Cell culture vessels (e.g., 6-well plates)
- Serological pipettes and various pipette capacities
- 70% ethanol
- Waste container
- Centrifuge machine (4°C)
- Vortex mixer
- TRIzol Reagent
- Chloroform
- Isopropanol
- RNase-free water
- Spectrophotometer and cuvettes or NanoDrop
Protocol Steps:
- Remove media from wells
- Add TRIzol reagent
- Incubate samples
- Homogenize samples.
- Add chloroform
- Shake vigorously using a vortex mixer.
- Incubate samples
- Centrifuge sample (12,000 x g, 15 minutes, 4°C)
- Carefully Transfer the aqueous phase to a new tube
- Add isopropanol; mix
- Bench incubation (for 10 minutes)
- Centrifuge sample (12,000 xg, 10 minutes, 4°C)
- Carefully aspirate supernatant; remove
- Add 70% ethanol; break the pellet using vortex
- Centrifuge (7500 x g, 5 minutes, 4°C)
- Carefully remove supernatant.
- Air-dry the RNA pellet
- Resuspend RNA pellet in a volume of RNase-Free water
- Measure the sample using a Nanodrop spectrophotometer.
Sample Reading:
- A protocol for determining the concentration and purity of the extracted RNA sample using spectrophotometry. This involves dilutions, blank creation, and absorbance measurements at specific wavelengths (260 nm, 280 nm, and 320 nm).
- Dilution factor calculation is essential.
- Blank preparation is important in ensuring accurate readings.
Spectrophotometer and Beer-Lambert Law: - The Beer-Lambert law describes the relationship between absorbance, path length, and concentration of a substance in a solution. - Absorbance is calculated from the ratio of initial light to final light intensity (A= log(Io/I)). - The Extinction coefficient is an important parameter in RNA analysis. It defines the molecule's ability to absorb light
Sample Quality Assessment:
- A method for assessing the purity and concentration of the RNA.
- The 260/280 ratio is used to assess protein contamination, the 260/230 ratio assesses salt contamination
- Optimal ratios for evaluating the RNA purity.
Questions:
- Basic steps of RNA extraction procedure.
- Determining the concentration of isolated human RNA.
- Potential experimental problems in the laboratory.
- Potential applications for this procedure in the medical field
RNA Concentration Calculation: A calculation method for determining the concentration of extracted RNA in µg/ml, considering impurities such as protein or salts.
RNA Quality Assessment: Methods to evaluate the purity of an RNA sample, including calculation ratios from spectrophotometry readings.

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Description

This quiz focuses on Lab Exercise 2 of the MD105 Cellular Biology course, emphasizing RNA extraction techniques and methodologies. It covers the theoretical aspects, protocols for RNA isolation, and principles of spectrophotometry related to sample quality assessment. Students will enhance their understanding of the challenges in RNA purification and the necessary equipment.