Molecular Biology Tuotorial--RNA Extraction RT-PCR

7/25/2019 Molecular Biology Tuotorial--RNA Extraction RT-PCR

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OUTLINE

DNA vs RNA (Comparison)

RNA extraction principles & procedures

Conventional PCR

Principle

Methodology

Applications

cDNA synthesis

Real-time PCR

Principle

Methodology

Applications


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DNA RNA

Stands for Deoxyribo Nucleic Acid. Ribo Nucleic Acid.

Function The blueprint of biological guidelinesthat a living organism must follow to

exist and remain functional.

Helps carry out DNA's blueprint

guidelines. Transfers genetic code

needed for the creation of proteins from

the nucleus to the ribosome

Structure Double-stranded.

G, C, A, T

Deoxy ribose sugar

Single-stranded.

G, C, A, U

Ribose sugar

Location DNA is found in the nucleus of a cell

and in mitochondria

Can be found in a cell's nucleus, its

cytoplasm, and its ribosome.

Stability More stable as the double helixminimizes full exposure to

enzymatic attack/degradation &

therefore decreases its degradation

Deoxy-ribose sugar is more stable

as it lacks one more OH group Degraded usually by Dnase enzyme

Less stable as the single stranded

chain is fully to enzymatic

attack/degradation & therefore it is

liable to its degradation

Ribose sugar with the extra 1 OH

group is more reactive and less stable Degraded usually by RNase H

enzyme

Propagation DNA is self-replicating Synthesized from DNA when needed

Unique

criteria

DNA is protected in the nucleus, as it

is tightly packed. DNA can bedamaged by exposure to ultra-violet

RNA is more resistant to damage by

Ultra-violet rays.


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RNAVS. DNA

RNA is degraded by RNase. This enzyme may be present onhands, mouth, airborne dust particles or laboratory glassware.It is difficult to inactivate RNase by autoclaving. Thereforevery rigorous conditions are required during RNA extractionto minimize RNAse activation. This requires wearing gloves,mask & decontaminating work place with DEPC (Di EthylPyro Carbonate) to degrade RNase enzyme.

On the other side, In DNA extraction, DNase that degradesDNA can be easily inactivated without the need for DEPC.

NB: Under acidic conditions, total RNA remains in the upperaqueous phase, while most of DNA and proteins remain eitherin the interphase or in the lower organic phase.


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WHY NEED RNA EXTRACTION

Detection of RNA viruses, degree of transcription

in cells by mRNA level.

Remember central dogma


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RNA EXTRACTION (PRINCIPLE & PROCEDURE)


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The nucleus is protected within a nuclear membrane which is also

surrounded by a cell membrane Lipid bilayer. Four steps are used

to remove and purify the RNA from the rest of the cell.

1. Lysis:Lysis is the disription/break of cell membranes to dissociate

nucleic acids from associated proteins and lipids and this would

allow the release of RNA that either found in nucleus or cytosol.

2. Removal of contaminants such as proteins, lipids &macromolecules by the use of chloroform.

3. Precipitation of nucleic acids by isopropanol or absolute

ethanol.

4. Washing (removal of residual/traces contaminants) by 70%

ethanol.

5. Resuspension of nucleic acids in TE (tris EDTA buffer) or

water.


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In principle; RNA extraction methodology has a very similar approach to

the Phenol:Chloroform method for DNA extraction except for the followings; .

In lysis step; In addition to phenol, we also use guanidine thiocyanatefor the following purposes

A- Cell memebrane disruption (Dissociation and rupture of lipid & protein

content).

B- Inactivation of Rnase enzyme

C- Dissociation of proteins from nucleoprotein complex.

Phenol used in this step is acidic with sodium acetate solution (Not neutral

as in DNA extraction.Why????)

No SDS detergent is used.

After addition of chloroform and centrifugation,

Total RNA remains in the

upper aqueous phase, while

most of DNA & proteins

remain either in the interphase

or in the lower organic phase.

RNA EXTRACTION (PRINCIPLE & PROCEDURE)


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CONVENTIONAL PCR (POLYMERASE

CHAIN REACTION)

PCR is a technique used to amplify DNA

molecule.

Application:Genetic analysis (Diseases)

Tissue typing for organ transplantation

Forensics


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PCR PRINCIPLE

Depend on using Thermocycler;

DenaturationForm ssDNA by high temp (95)

AnnealingHybridization between primer and ssDNA.

Elongation

DNA starts building complementary strand by

using available nucleotides.


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Steps of PCR:

1- Initial step: heating the reaction at 95C for 1-10 minutes to activate

the DNA polymerase and also to denature the 2 strands

(unwinding=separating).

2- Several consecutive cycles (20-50) are operated. Each cycle includes

three basic steps;

A- Denaturation: Where the two strands of DNA will be separated athigh temperature (95C) through the destruction of hydrogen bonds

(A=T & GC).

B- Annealing: Where two oligonucleotide primers (22-30 nuncleotide

bases each) bind to their complimentary sequences on the DNA template.

These primers are short single stranded oligonucleotides.

C- Extension: where Taq DNA polymerase starts to add complementary

dNTPs (based on template DNA sequence) to form new strands.


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Taq DNA polymerase adds nucleotides in the 5---3 direction only.

3- Final extension: This is a single and final step that is performed after

repetitive cycles (20-50) to ensure that all the newly amplified

fragments are fully elongated with correct base insertion as TaqPolymerase adds nucleotides very quickly with a possibility for either

incorporationg wrong nucleotides or even missing the addition of

certain nucleotides. This final step would enable Taq polymerase to

correct these nucleotides through 3----5 proof reading criteria.

NB: The products of PCR reactions are then analyzed by separation on

agarose gels followed by ethidium bromide staining and visualization

with uv transillumination.


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PCR STEPS


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PCR FOR RNA?!

PCR uses only DNA. So how can we amplifyRNA?

By synthesizing a cDNA from the RNA, by using

Reverse transcriptase enzyme. Degrading the RNA by RNase.

Form dsDNA

After formation of cDNA, you can perform PCR.


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CDNASYNTHESIS

What nucleotides we should add?????


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GENE EXPRESSION

Remember central dogma

Amount of mRNA reflect the gene expression.

To quantify mRNA by PCR, first mRNA cDNA

(used as template in PCR).


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REAL-TIME PCR

Its a modified PCR technique

Used to monitor the progress of PCR, quantifying

the amplified DNA during reaction.

Quantitative PCR (qPCR). No gel-based analysis at the end of the PCR

reaction.

Computer based analysis of fluoresence.


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PRINCIPLE

By utilizing a substance (Flurophore) the will

only bind to the dsDNA molecule

By increasing the number of cycles, more product

will be formed, consequently more signal will be

measured.


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PROCEDURE

Methodology used similar to conventional PCR.

Material used

DNA template

pair of specific primers

dNTPs Buffer solution

Taq polymerase

Substance marked with a fluorophore named asProbe.

Steps

Denaturation

Annealing

Extension


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PRODUCT DETECTION

Depending on

fluoroesnce of probes.

Control

Cycle threshold


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PROBE TYPES

Syber Green:Binds to only dsDNA, non specific

Taqman:Composed of a flucophore and a quencher on the

same molecule. Flucophore emission is preventedby the close vicinity of the quencher. Afterdegradation, emission will increase. Highlyspecific, as it depends on complementarity withtemplate.

Molecular beacon:single stranded hairpin shaped oligonucleotideprobes. In the presence of the target sequence,they unfold, bind and fluoresce upon excitation.


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PROBE TYPES


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CONTROL

Negative: to assure no contamination of reaction

reagents.

Positive: to assure the quality of reagents use,

e.g: primers anneal to target region.

HKG (House Keeping Gene): to compare between

your desired gene and other genes that are

expressed normally in each cell.


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CYCLE THRESHOLD

The Ct (cycle threshold) is defined as the number

of cycles (PCR cycles) required for the fluorescent

signal to cross the threshold (i.e exceeds noisy

background level).

As Ct value increases, this means that the

original starting RNA is low= low expressed RNA


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CYCLE THRESHOLD


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CYCLE THRESHOLD


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QPCRAPPLICATIONS:

Gene expression analysis

MicroRNA analysis

Single nucleotide polymorphism (SNP)

genotyping

Molecular Biology Tuotorial--RNA Extraction RT-PCR

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