Lab 3: isolation of RNA and analysis of gene expression by RT-PCR

Question 1

RNA

Answer 1

Long polymer of nucleoside monophosphate linked by phosphodiester bonds.

Question 2

Chemical differences between RNA & DNA

Answer 2

• RNA has ribose sugar with a hydroxyl group (-OH) at 2’ carbon; DNA has a deoxyribose (no OH at 2’ carbon).
• RNA uses Uracil (U) instead of thymine (T) at the nitrogenous base.

-RNA is single stranded.

• The hydroxyl group at 2’ carbon makes RNA sensitive to degradation under mildly elevated pH and temp.
• RNA isolation can be fragile due to its fragile nature compared to DNA -> can be used to measure gene expression by analyzing mRNA levels.

Question 3

What are some previous models to measure Gene Expression?

Answer 3

• Northern blotting
• RNAse protection assays

** These are time consuming and need radioactive probes.

Question 4

What is the modern method to measure Gene Expression?

Answer 4

• Real-Time PCR (RT-PCR), couples RT (reverse transcriptase) & PCR to rapidly analyze gene expression.
• RT converts RNA to cDNA.

Question 5

OligodT primer

Answer 5

• used to initiate the reverse transcription reaction.
• binds to polyA tails of mRNA, allowing RT to make cDNA.

Question 6

What is the focus of this experiment?

Answer 6

To study gene expression patterns of p53-null and WT human HCT116 codon cancer cells.

-> P53 gene (most frequently mutated in cancer; mutation leads to resistance to chemotherapy (eg., 5-FU).

-> p53 activates p21 - which inhibits cell cycle progression.

This experiment measures p21 expression in response to 5-FU treatment.

Question 7

In week 1, two main steps took place (1) RNA Isolation and (2) cDNA synthesis. There are p53-null (mutated p53) and WT (normal p53 cells). Cells will be treated with 5-FU (chemotherapy agent) or left untreated. Describe the process of step (1) and (2).

Answer 7

(1)
RNA isolation
- RNA purification:
Isolate total RNA from each patient.
Quantify RNA using spectrophotometer.
Assess RNA quality using an agarose gel.

(2)
First-strand cDNA synthesis.
- Use RT mRNA -> cDNA
- OligodT primers.

Question 8

Chloroform

Answer 8

Separates cellular contents into phases during extraction, chloroform helps RNA into aqueous phase, removes proteins & lipids.

Question 9

Isopropanol

Answer 9

Precipitates RNA by removing it from solution.

Question 10

Ethanol

Answer 10

Washes/removes contaminants.

Question 11

RNAse-free water

Answer 11

Dissolves RNA pellet after ethanol wash to prepare it for quantification and further use.

Question 12

Trizol

Answer 12

• disrupts macromolecules to release RNA from cells.

Question 13

Guanidium Thiocynate

Answer 13

Denatures RNases, protecting RNA from degradation.

Question 14

Is Phenol a hazard or no?

Answer 14

Yes!
Corrosive.
Warning.

Question 15

In week 2, you perform PCR analysis. What are the two types that you perform, what are the differences between them and the processes of each.

Answer 15

(1) End-point PCR:
- amplify specific DNA sequence from DNA template.
- used to confirm the presence or absence of gene of interest and to visualize the amplification of products.
- on the cDNA from week 1.
- measure p21 expression as major target.
- use GAPDH as housekeeping gene for comparison.
- compare treated vs untreated cells, p53-null & WT cells - gene expression.

(2) Real-time pCR (qPCR)
- detect and quantify RNA.
- quantitative assessment of gene expression by measuring amount of PCR product generated in real-time during each cell.

Question 16

What was the purpose of water and Mg2+ being used?

Answer 16

• Water was used as a solvent.
• Mg2+ was required for activity of Taq polymerase enzyme.

Question 17

What was the cDNA template diluted to for qPCR and why?

Answer 17

• 1/80 cDNA dilution.
• to reduce cDNA concentration for qPCR analysis, ensuring accurate quantification of gene expression.

Question 18

What will a melting curve analysis tell us?

Answer 18

• monitors product specificity by measuring temperature a which DNA dissociates (melts) during final cycle.

Question 19

What should the result of this experiment be?

Answer 19

• WT p53 cells treated with 5FU - higher expression of p21 compared to untreated WT/
• 5FU activates p53 expression -> bind to p21 and increase transcription of p21 (cyclin dependent kinase inhibitor, slows cell cycle progression, inhibit cancer growth)
• in p53-null, there is minimal increase in p21.
• when RNA was extracted from 4 samples, showed bands at 3311, 1995, 139 bases (28S, 18S, 5S rRNA) showing successful RNA extraction.