MD105 Lab Exercise 2: RNA Extraction

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Questions and Answers

What occurs during centrifugation at 12,000 x g for 15 minutes at 4°C?

Mixed particles remain uniformly distributed.

Denser particles settle to the bottom while less dense substances remain at the top. (correct)

All materials are turned into a homogenous solution.

Low-density substances settle at the bottom.

What is the purpose of adding chloroform to the homogenate?

To create phase separation of substances. (correct)

To stabilize the RNA during centrifugation.

To enhance DNA recovery.

To promote RNA degradation.

What do you observe when the aqueous phase is carefully transferred to a new tube?

No visible changes occur. (correct)

The RNA is typically visible as a gel-like or white pellet.

Visible protein aggregates.

The sample becomes a clear liquid.

What does the dilution factor for a 20/1000 dilution result in?

1:100 Signup and view all the answers

Why is it necessary to read absorbance at both 260nm and 320nm?

To assess protein contamination impurities. Signup and view all the answers

What is the result of adding 70% ethanol before centrifugation at 7500 x g?

Precipitates RNA from the solution. Signup and view all the answers

What must be done with the supernatant after centrifugation when extracting RNA?

It should be carefully aspirated and discarded. Signup and view all the answers

What is the significance of breaking the RNA pellet before the final centrifugation?

To enhance the quality of RNA extraction. Signup and view all the answers

What is the purpose of measuring absorbance at 260 nm for nucleic acid assessment?

To determine RNA or DNA concentration Signup and view all the answers

Which value indicates a problem with RNA purity?

260/280 ratio of less than 1.7 Signup and view all the answers

What does the Beer-Lambert Law relate to in the context of RNA concentration measurement?

Absorbance to concentration and path length Signup and view all the answers

What is the optimal 260/280 ratio indicating high purity in RNA samples?

2.0 Signup and view all the answers

Why is it important to measure absorbance at 230 nm?

To evaluate salt contamination Signup and view all the answers

Which absorbance ratio is typically used to assess salt contamination in RNA samples?

260/230 Signup and view all the answers

What is the typical absorbance at 320 nm used for during RNA quality assessment?

Identifying dirty cuvette issues Signup and view all the answers

What does the extinction coefficient (ε) represent in Beer-Lambert Law?

The specific ability of molecules to absorb light Signup and view all the answers

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

To maintain RNA integrity during cell homogenization Signup and view all the answers

Why is careful handling and the use of RNase-free solutions important in RNA extraction?

RNases degrade RNA and affect sample quality Signup and view all the answers

What is the correct procedure after adding TRIzol Reagent to the cells in a 6-well plate?

Leave the sample at the bench for 10 minutes Signup and view all the answers

Which methods are classified as cell lysis methods in RNA extraction?

Physical and reagent-based methods Signup and view all the answers

To ensure high quality RNA extraction, what condition is essential during cell incubation?

Optimal temperature at 37.0°C and humidity above 90% Signup and view all the answers

Which equipment is necessary for assessing the quality of RNA samples?

NanoDrop or spectrophotometer Signup and view all the answers

What role does isopropanol play in RNA extraction?

It is used for RNA precipitation Signup and view all the answers

What is a potential consequence of not maintaining RNase-free conditions during RNA extraction?

Degradation of the RNA sample Signup and view all the answers

Study Notes

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

Course: MD105 - Cellular Biology Laboratory
Lab Exercise: RNA extraction, quantification, and sample quality assessment
Semester: Fall 2024
Objectives:
- Understand the theoretical background of RNA extraction, including introduction, cell lysis, and purification.
- Learn the methodology and protocol for RNA extraction using TRIzol, precipitation with isopropanol, and purification with ethanol.
- Understand spectrophotometry principles for sample quality assessment.
Introduction to RNA Extraction:
- Ribonucleic acid (RNA) is a crucial biological macromolecule essential for life, alongside DNA and proteins.
- RNA extraction is the process of purifying RNA from biological samples.
- Isolating intact RNA can be challenging due to the high abundance of RNases (enzymes that degrade RNA).
- Proper technique, including careful sample handling, aseptic techniques, and RNase-free solutions, is crucial during extraction.
- High-quality and -quantity RNA extraction from monolayer cells is essential for gene expression studies.
Cell Lysis:
- Methods for cell lysis include reagent-based and physical methods.
- The lab uses TRIzol reagent, a monophasic solution of phenol and guanidine isothiocyanate.
- TRIzol maintains RNA integrity during homogenization while disrupting cells and components.
- It is commonly used for adherent cell detachment.
Materials and Equipment:
- Cell culture hood
- Cell incubator (37°C, >90% humidity, 5% CO2)
- Cell culture vessels (e.g., 6-well plates)
- Pipettes (various capacities)
- 70% ethanol
- Waste container
- Centrifuge (4°C)
- Vortex mixer
- TRIzol reagent
- Chloroform
- Isopropanol
- RNase-free water
- Spectrophotometer (and cuvettes or NanoDrop)

Protocol

Step 1: Remove media from 6-well plate wells, add 0.5ml of TRIzol reagent to each well, and leave for 10 minutes.
Step 2: Homogenize the sample and transfer to a tube. Leave at room temperature for 5 minutes.
Step 3: Add 0.2ml of chloroform, cap, vortex for 15 seconds and let sit 2-3 minutes
Step 4: Centrifuge at 12,000 x g for 15 minutes at 4°C. Observe what happens to the sample.
Step 5: Transfer the upper (aqueous) phase to a new tube. (Why?)
Step 6: Add 0.5ml isopropanol and thoroughly vortex the new tube.
Step 7: Incubate the tube at room temperature for 10 minutes.
Step 8: Centrifuge at 12,000 x g for 10 minutes at 4°C.
Step 9: Carefully remove and discard the supernatant.
Step 10: Add 0.5ml of 70% ethanol, break the pellet, and centrifuge at 7500 x g for 5 minutes at 4°C.
Step 11: Remove the supernatant completely, and briefly air-dry the RNA pellet.
Step 12: Redissolve the RNA in an appropriate volume of RNase-free water.
Step 13: Measure the sample using a nanodrop spectrophotometer.

Sample Reading

Step 1: Dilute RNA sample (20/1000 final volume, 2500µl dH2O). Calculate the dilution factor.
Step 2: Transfer dilution to a separate cuvette.
Step 3: Prepare a blank cuvette with 2500 µl dH2O.
Step 4-6: Adjust and blank the spectrophotometer at 260nm. Measure and record the absorbance of the diluted RNA sample at 260nm; and any impurities read absorbance at 320nm for further assessment, record values

Sample Quality Assessment and Purity

Important ratios: A260/A280, A260/A230.
Purpose: Assess RNA purity and concentration.
Optimal Ratios:
- 260/230 for RNA should be 2.00 (range: 2.00 - 2.20.)
- 260/280 for RNA should be 2.00 (range: 1.80 - 2.00).

Determining RNA Concentration and Purity

260/230 and 260/280: These ratios indicate the concentration of nucleic acids, proteins, and salts in the sample.
260 nm: Nucleic acids absorb at 260nm.
280 nm: Proteins absorb at 280nm.
320 nm: Assess for impurities and debris/background.

Concentration Calculation

Formula: RNA concentration (µg/ml) = [(A260 – A320)/0.025] x Dilution Factor; Total RNA (µg) = RNA concentration (µg/ml) x final sample volume (ml).

Quality assessment of the RNA sample

Formulaes:*
A260/A280 = (A260 – A320) ÷ (A280 - A320)
A260/A230 = (A260- A320) ÷ (A230- A320)

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Description

This quiz focuses on MD105 Cellular Biology Laboratory's second lab exercise, which covers RNA extraction techniques, quantification, and quality assessment. Participants will learn about the theoretical background of RNA extraction, methodologies using TRIzol, and principles of spectrophotometry for sample evaluation.