Lab 3 (Isolation of RNA and Analysis of Gene Expression by RT-PCR) Flashcards
(28 cards)
What are the objectives of this lab?
1) Isolate total RNA from cultured human cells.
2) Convert mRNA into cDNA using reverse transcription.
3) Analyse gene expression using endpoint and real-time PCR (qPCR).
Background for this Lab
Lab 3 focuses on studying gene expression by analysing mRNA levels. Traditionally, techniques like Northern blotting and RNase protection assays were employed, which were time-consuming and required radioactive probes. However, advancements in real-time PCR technology allow for a more sensitive and rapid analysis by combining reverse transcription with PCR (RT-PCR).
Before PCR can be used to analyse mRNA levels, the RNA must first be converted into cDNA using the enzyme reverse transcriptase (RT). This enzyme requires a primer to start the process, often an oligonucleotide composed of 18-20 deoxythymidines (oligo dT).
The oligo dT binds to the poly(A) tails of mRNA molecules, and the reverse transcriptase then synthesizes a complementary DNA strand (cDNA) based on the mRNA template. This process is referred to as first-strand cDNA synthesis.
Summary of this Experiment (Week 1 and 2)
In Lab 3, you will purify RNA from cultured human HCT116 colon cancer cells and compare gene expression patterns between cells with and without functional p53. The cells are treated with the chemotherapy agent 5-fluorouracil (5-FU) or left untreated. The experiment aims to measure the effect of p53 on the induction of the p21 gene in response to 5-FU treatment.
1) Week 1: Isolate and purify total RNA from two types of colorectal cancer cells, both treated and untreated. Quantify the RNA spectrophotometrically and assess its quality on an agarose gel. Perform first-strand cDNA synthesis using reverse transcriptase.
2) Week 2: Perform endpoint and real-time PCR analysis on the cDNA to analyse p21 expression, comparing it to the expression of the housekeeping gene GAPDH.
How is RNA Isolated?
RNA is more prone to degradation than DNA due to its chemical structure and the presence of ubiquitous RNases. Therefore, careful handling and specific techniques are crucial for successful RNA isolation.
Guanidinium thiocyanate-phenol-chloroform extraction is a common method used to purify RNA. This method utilizes guanidinium thiocyanate, a chaotropic agent, which denatures proteins, including RNases, protecting RNA from degradation. Phenol and chloroform are organic solvents used to separate the RNA from DNA and proteins. The RNA remains in the aqueous phase, while DNA and proteins partition into the organic phase.
RNase-free Water
Used throughout the experiment to prevent RNase contamination and degradation of RNA.
Lysis Solution
Contains guanidinium thiocyanate to lyse cells and denature RNases, protecting RNA.
Chloroform
An organic solvent used to separate RNA from DNA and proteins during extraction.
Isopropanol
Precipitates RNA from the aqueous phase
70% Ethanol
Washes the RNA pellet to remove residual salts and contaminants.
TBE Buffer
Used for agarose gel electrophoresis to seperate and visualise RNA.
RNA Loading Dye
Contains formamide to denature RNA and tracking dyes to visualise RNA migration during electrophoresis.
Oligo (dT)19 Primer
Binds to the poly(A) tail of mRNA, providing a starting point for reverse transcriptase.
dNTP Mix
Building blocks (dATP, dCTP, dGTP, dTTP) for cDNA synthesis.
MuLV Reverse Transcriptase
An RNA-dependent DNA polymerase that synthesises cDNA from the mRNA template.
5X RT Reaction Buffer
Provides optimal conditions (pH, salts) for reverse transcriptase activity.
PCR Buffer
Provides optimal pH and salt conditions for DNA polymerase activity during PCR.
Forward and Reserve Primers
Short, single-stranded DNA sequences that are specific to the target genes (GAPDH and p21) and are used to amplify the cDNA during PCR.
Taq DNA Polymerase
A thermostable DNA polymerase that amplifies DNA during PCR.
MgCL2
Acts as a cofactor for DNA polymerase.
2X qPCR Mix
Contains a thermostable DNA polymerase, dNTPs, and a fluorescent dye (SYBR Green I) for qPCR.
SYBR Green I Dye
A fluorescent dye that binds to double-stranded DNA and is used to detect and quantify PCR products in real-time.
DNA Ladder
A set of DNA fragments of known sizes, used as a reference to determine the size of the PCR products on an agarose gel.
Why is RNA more susceptible to degradation than DNA? How can RNA degradation be prevented during isolation?
RNA is more prone to degradation than DNA due to the presence of a 2’-hydroxyl group on the ribose sugar, which makes it chemically more reactive. Also, RNases, enzymes that degrade RNA, are ubiquitous and highly stable. To prevent RNA degradation during isolation, the following precautions are taken:
1) Use RNase-free reagents and equipment: All reagents and materials used should be certified RNase-free.
2) Work quickly and on ice: Keeping the samples cold slows down enzymatic activity.
3) Use a chaotropic agent: Guanidinium thiocyanate in the lysis solution denatures RNases, rendering them inactive.
4) Wear gloves: Gloves prevent RNase contamination from hands.
What is the purpose of reverse transcription in this experiment? Describe the steps involved in first-strand cDNA synthesis.
Reverse transcription is necessary to convert mRNA into cDNA, which is more stable and suitable for PCR amplification. The steps involved in first-strand cDNA synthesis are:
1) Annealing: The oligo (dT)18 primer anneals to the poly(A) tail of the mRNA.
2) Extension: Reverse transcriptase uses the mRNA as a template and the dNTPs as building blocks to synthesize a complementary DNA strand (cDNA).
3) Inactivation: The reaction is heated to inactivate the reverse transcriptase enzyme.
