MD105 - Cellular Biology Lab RNA Extraction

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

What should be added to the homogenate after waiting for 5 minutes at room temperature?

0.5 ml isopropanol

dH2O

0.2 ml chloroform (correct)

70% ethanol

During centrifugation at 12,000 x g, which phase contains RNA after phase separation?

Interphase

Organic phase

Aqueous phase (correct)

Sediment phase

What is a critical precaution to take before centrifuging your sample?

Cap the tubes tightly

Ensure the sample is completely thawed

Cool the sample to room temperature

Make sure the centrifuge is balanced (correct)

What is the purpose of adding isopropanol to the sample?

To precipitate the RNA Signup and view all the answers

In the context of diluting a sample, what does a dilution factor of 20/1000 indicate?

A final volume of 2500 μl Signup and view all the answers

At which wavelength should absorbance be measured for assessing protein contamination?

280 nm Signup and view all the answers

What is the appearance of the RNA pellet after centrifugation?

Gel-like or white Signup and view all the answers

What should you do with the supernatant after aspirating it from the RNA pellet?

Discard it completely Signup and view all the answers

What is the primary challenge in isolating intact RNA from biological samples?

The presence of RNases that degrade RNA molecules Signup and view all the answers

Which method is used in the laboratory protocol for lysis of cells?

TRIzol Reagent application Signup and view all the answers

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

It helps in the precipitation of RNA Signup and view all the answers

What is the purpose of using RNase-free solutions during RNA extraction?

To prevent RNA degradation during extraction Signup and view all the answers

What type of sampling conditions are ideal for cell culture before RNA extraction?

Optimal temperature of 37.0°C with high humidity Signup and view all the answers

Which of the following statements about cell lysis methods is true?

Reagent-based methods use formulated lysis buffers to disrupt cell membranes Signup and view all the answers

What equipment is essential for measuring the quality of extracted RNA?

Spectrophotometer or NanoDrop Signup and view all the answers

Which reagent is specifically mentioned as maintaining RNA integrity during homogenization?

TRIzol Reagent Signup and view all the answers

What should the 260/280 absorbance ratio be for optimal purity of RNA?

Around 2.0 Signup and view all the answers

What is the significance of measuring absorbance at 230 nm in the RNA purification process?

It assesses salt contamination. Signup and view all the answers

According to the Beer-Lambert Law, absorbance (A) is dependent on which of the following factors?

Path length, concentration, and absorptivity Signup and view all the answers

What is indicated by a 260/230 absorbance ratio of 2.0 to 2.2?

Optimal purity concerning salt contamination Signup and view all the answers

If the absorbance at 320 nm is high, what does that suggest?

Contamination due to extraneous debris. Signup and view all the answers

How is the concentration of RNA in ng/μl calculated?

By using the equation A = ε L C and knowing the absorbance. Signup and view all the answers

Which absorbance values are primarily used to assess the purity of DNA and RNA?

260 nm and 280 nm Signup and view all the answers

To what does the term 'ε' in the Beer-Lambert Law refer?

The absorptivity or extinction coefficient of specific molecules. Signup and view all the answers

Study Notes

MD105 - Cellular Biology 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

Theoretical Background: Introduction to RNA extraction, cell lysis and purification, materials and equipment.
Methodology and Protocol: RNA isolation and separation using TRIzol, RNA precipitation using isopropanol, RNA purification using ethanol, spectrophotometry principles for sample quality assessment.

Introduction to RNA Extraction

Ribonucleic acid (RNA) is one of the three major biological macromolecules essential for life, alongside DNA and proteins.
RNA extraction is the purification of RNA from biological samples.
Isolating intact RNA is challenging as RNases, enzymes that degrade RNA, exist abundatly in the environment, making complete removal or destruction difficult.
RNA isolation requires careful handling, good aseptic technique and RNase-free solutions.
Extracting high-quality RNA from monolayer cells is critical for many gene expression experiments.

Cell Lysis: Reagent-Based

Cell lysis methods include reagent-based and physical methods.
Reagent-based methods use specific lysis buffers to disrupt cell membranes.
TRIzol reagent, a mono-phasic solution of phenol and guanidine isothiocyanate, maintains RNA integrity during homogenization, while simultaneously disrupting cells and their components.
TRIzol reagent is suitable for detaching and processing adherent cells and is available commercially.

Materials and Equipment

Cell culture hood
Cell incubator (optimal temperature 37.0°C, humidity >90%, CO2 5%)
Cell culture vessels (e.g., 6-well plates)
Serological pipettor and various pipettes
70% ethanol
Waste container
Centrifuge machine (set at 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 and add 0.5ml of TRIzol reagent to each well. Incubate samples for 10 minutes at room temperature.
Homogenize the sample.
Add 0.2ml chloroform, cap the tube, and vortex for 15 seconds.
Incubate the sample at room temperature for 2-3 minutes.
Centrifuge at 12,000 x g for 15 minutes at 4°C.

Centrifugation

Ensure the centrifuge is balanced properly; unbalanced loads can lead to instability and inaccurate results.
Centrifugation separates substances based on density, with denser substances sedimenting at the bottom.

Phase Separation

After centrifugation, the sample will separate into phases. The RNA will reside in an aqueous upper layer.
Carefully transfer the aqueous phase to a new tube.

Protocol (Steps 7-14)

Add 0.5ml of isopropanol to the aqueous phase and vortex.
Incubate the solution at room temperature for 10 minutes.
Centrifuge at 12,000 x g for 10 minutes at 4°C
Remove the supernatant. The RNA pellet will be visible at the bottom of the tube as a gel-like or white mass.
Add 0.5ml of 70% ethanol, break the pellet, and centrifuge at 7500 x g for 5 minutes at 4°C Remove the supernatant completely and allow the RNA pellet to air dry briefly.
Resuspend the RNA pellet in an appropriate volume of RNase-free water (e.g., 30µl).
Measure the sample using a NanoDrop spectrophotometer.

Sample Reading

Dilution: Dilute RNA samples with a known concentration and calculate the dilution factor.
Spectrophotometry: Use a spectrophotometer to measure the absorbance of diluted RNA at different wavelengths (260nm, 280nm, 230nm, 320nm). Record the absorbance values.

Sample Quality Assessment

Concentration: The concentration calculation involves the absorbance measurements at 260nm after considering impurities
Purity: Evaluate RNA purity by calculating absorbance ratios, such as 260/280 and 260/230
Optimal Ratios: Target 260/280 and 260/230 ratios for RNA, that can be indicators of purity problems.

Questions

Explain the four basic steps of RNA extraction.
Explain how to determine the concentration of isolated human RNA.
List potential laboratory issues during this experiment.
Describe potential applications of this procedure in the medical field.

Determination of RNA concentration

Calculation of RNA concentration using absorbance at 260nm, corrected for impurities at 320nm, and considering the dilution factor.

Quality Assessment of RNA

Determination of RNA purity using absorbance ratios (260/280 and 260/230).

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

Description

This quiz explores the key concepts involved in RNA extraction, as outlined in the MD105 Cellular Biology Laboratory course. It covers RNA quantification, sample quality assessment, and the methodologies for isolating RNA, including the use of TRIzol and spectrophotometry. Test your knowledge on this essential biological process.