MD105 Cellular Biology Lab Exercise 2: RNA Extraction

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Questions and Answers

What is the primary purpose of RNA extraction?

To degrade RNA molecules for disposal

To isolate DNA from cells

To purify RNA for gene expression studies (correct)

To precipitate proteins for analysis

Which reagent is specifically mentioned as a crucial component for cell lysis in RNA extraction?

Chloroform

Ethanol

Isopropanol

TRIzol Reagent (correct)

What type of methods are mentioned for cell lysis during RNA extraction?

Biochemical and mechanical methods

Reagent-based and physical methods (correct)

Chemical and thermal methods

Enzymatic and suppression methods

Why is it important to handle RNA extraction samples with caution?

To avoid contamination and degradation by RNases Signup and view all the answers

Which of the following is NOT a required piece of equipment for RNA extraction?

Microcentrifuge tubes Signup and view all the answers

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

To precipitate RNA Signup and view all the answers

What does the use of RNase-free solutions ensure during RNA extraction?

Preservation of RNA integrity Signup and view all the answers

Which environmental condition is crucial for the operation of a cell incubator used in RNA extraction?

Maintaining >90% humidity Signup and view all the answers

What is the purpose of adding chloroform to the homogenate?

To initiate phase separation of components Signup and view all the answers

What will occur if the centrifuge is not properly balanced during the procedure?

The sample will not separate correctly Signup and view all the answers

In the phase separation process after centrifugation, where is RNA found?

In the aqueous phase Signup and view all the answers

What is the expected observation after aspirating the supernatant post-centrifugation?

A visible gel-like or white pellet at the bottom Signup and view all the answers

What is the primary reason for adding isopropanol after transferring the aqueous phase?

To precipitate RNA Signup and view all the answers

What is the dilution factor when preparing a diluted RNA sample of 2500μl from a concentration of 20/1000?

$1/5$ Signup and view all the answers

Which absorbance wavelength is used to assess protein contamination in the RNA sample?

280 nm Signup and view all the answers

What is the recommended final volume when diluting an RNA sample with water?

2500μl Signup and view all the answers

What is the significance of the 260/280 absorbance ratio in RNA quality assessment?

Assesses the purity of RNA with respect to proteins Signup and view all the answers

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

Possible contamination of RNA by proteins Signup and view all the answers

According to Beer-Lambert Law, which of the following factors does NOT influence absorbance (A)?

Type of cuvette used Signup and view all the answers

What is the ideal absorbance value range at 260nm to indicate a pure sample of RNA?

1.8 - 2.0 Signup and view all the answers

When measuring absorbance at 230nm, which of the following is it primarily used to assess?

Presence of salt contaminants Signup and view all the answers

What does a 260/230 absorbance ratio of approximately 2.0 indicate?

Low protein contamination in the RNA sample Signup and view all the answers

Which statement about measuring absorbance at 320nm is accurate?

It measures background noise or debris in the sample. Signup and view all the answers

How can the concentration of isolated RNA be determined?

Using the equation C = A/ε L 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 of RNA and sample quality assessment
Semester: Fall 2024
Objectives: 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 three major biological macromolecules crucial for all life, along with DNA and proteins.
RNA extraction is the purification of RNA from biological samples.
Isolating intact RNA is challenging because RNases (RNA-degrading enzymes) are prevalent in the environment, making complete removal/destruction difficult.
RNA isolation requires careful sample handling, aseptic technique, and RNase-free solutions.
Extracting high-quality, abundant RNA from cells is essential for gene expression studies.

Cell Lysis: Reagent-based

Cell lysis methods involve reagents or physical means.
Reagent-based lysis uses specific buffers to disrupt cell membranes.
TRIzol reagent disrupts and breaks down cells and cell components while maintaining RNA integrity during homogenization.
TRIzol is a mono-phasic solution of phenol and guanidine isothiocyanate, for adherent cell detachment.

Materials and Equipment

Cell culture hood
Cell incubator (37.0°C, >90% humidity, 5% CO2)
Cell culture vessels (6-well plates)
Serological pipettors and pipettes
70% ethanol
Waste container
Centrifuge machine (4°C)
Vortex
TRIzol reagent
Chloroform
Isopropanol
RNase-free water
Spectrophotometer and cuvettes or NanoDrop

Protocol (Steps 1-5)

Remove media from 6-well plates containing samples.
Add 0.5ml of TRIzol reagent to each well and incubate for 10 minutes.
Transfer homogenate to a tube.
Incubate the tube for 5 minutes at room temperature.
Add 0.2ml of chloroform to the tube. Vortex for 15 seconds.
Incubate the tube at room temperature for 2-3 minutes.
Centrifuge at 12,000 x g at 4°C for 15 minutes.

Centrifugation

Unbalanced centrifuges are unstable and do not spin correctly.
Denser particles settle at the bottom, while low-density materials rise to the top.

Phase Separation

The mixture separates into aqueous phase (RNA), interphase (DNA), and organic phase (protein/lipids).
Carefully transfer the aqueous phase to a new tube.

Protocol (Steps 6-14)

Add 0.5ml of isopropanol and mix using vortex.
Incubate the tube at room temperature for 10 minutes.
Centrifuge at 12,000 x g at 4°C for 10 minutes.
Aspirate and discard the supernatant.
Add 0.5ml of 70% ethanol, break the RNA pellet, and centrifuge at 7500 x g for 5 minutes at 4°C.
Remove supernatant. Briefly air-dry the RNA pellet.
Redissolve the pellet in an appropriate volume of RNase-free water (30µL).
Measure the sample at a nanodrop spectrophotometer.

Sample Reading (Steps 1-6)

Dilute RNA sample (20/1000 with dH2O, final volume 2500µl). Calculate dilution factor.
Mix and transfer diluted sample to a cuvette.
Transfer 2500µl of dH₂O to a separate cuvette for blank.
Adjust spectrophotometer to 260nm and blank with dH₂O.
Measure absorbance of diluted RNA sample at 260nm. Note absorbance value
Measure absorbance at 320nm to evaluate sample purity (impurities).

Sample Reading (Steps 7-12)

Adjust spectrophotometer to 320nm and create a blank using dH₂O.
Measure absorbance of the RNA dilution at 320nm. Write down absorbance value.
Protein contamination assessment:
Adjust spectrophotometer to 280nm and blank it.
Measure the absorbance of diluted RNA at 280nm
Write down the absorbance value for each dilution.
Salt contamination assessment:
Adjust spectrophotometer to 230nm and blank it.
Measure the absorbance of diluted RNA at 230nm.
Write down the absorbance value for each dilution.

Spectrophotometer - Beer-Lambert Law

Absorbance depends on path length (L) and concentration.
Extinction coefficient values are shown for different molecules (e.g., double-stranded DNA, single-stranded RNA, single-stranded DNA).
Formula provided for calculating concentration

Sample Quality Assessment - Analysis

DNA/RNA peak at 260nm.
Proteins absorb at 280nm.
Salt contamination at 230nm
Absorbance ratio 260/280 and 260/230 determine purity
260/280 ~1.8 in DNA, ~2 in RNA
260/230 ~2.0 to 2.2
Absorbance at 320nm helps to adjust other wavelength readings (background subtraction).

Sample Quality Assessment - Measurement

Concentration & purity in ng/µl
Purity determination using ratios (260/280, 260/230) for protein and salt, respectively
Optimal ratios: for RNA, 2.0 -2.2 at 260/230, and 1.80-2 at 260/280

Questions (from the document)

What are the four basic steps of RNA extraction?
How do you determine the concentration of isolated human RNA?
List any lab problems in performing this experiment.
Can you think of medical fields that use this procedure?

RNA Concentration Calculation

RNA concentration (µg/ml) = [(A260 - A320)/0.025] x DF
Total RNA amount (µg) = RNA concentration (µg/ml) x final sample volume (mL)

RNA Sample Quality Assessment

260/280 = (A260 - A320) / (A280 - A320)
260/230 = (A260 - A320) / (A230 - A320)

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Description

This quiz covers the key concepts and methodologies involved in RNA extraction as part of the MD105 Cellular Biology Laboratory course. Students will learn about cell lysis, purification techniques, and the principles of spectrophotometry for assessing sample quality. Prepare to test your understanding of RNA extraction methods and protocols.