Analysis of gene expression using RT-PCR

Dr. Tamas Borsics

ABE Workshop 2006 Leeward Community College

9:30 am Friday, June 16, 2006

What does the term “RT-PCR” stand for?

Involves two processes:

RT – Reverse Transcription

During this step we synthesize single stranded DNA from RNA template

PCR – Polymerase chain reaction

Using gene-specific primers we amplify a certain part of our gene of interest to get enough amount for further analysis

DNA: Double stranded – very stable, durable

• Used in cells to store information (chromosomes)

• Material of inheritance

• Used in forensic science

• Used by archeologists (~ 50.000 years)

DNA and RNA – What a difference!

RNA: single stranded – unstable, strong secondary structures

• Used in cells to temporary express information

• Cell’s RNA profile changes from minute-to-minute

• Degrades very easily – even during the isolation process

DNA and RNA – What a difference!

The DNA replicates its information in a process that involves many enzymes

The DNA codes for the production of messenger RNA (mRNA)

In eucaryotic cells, the mRNA is processed (essentially by splicing) and migrates from the nucleus to the cytoplasm

Messenger RNA carries coded information to ribosomes. The ribosomes "read" this information and use it for protein synthesis

The Central dogma of Molecular BiologyU

NID

IRE

CT

ION

AL

What makes the difference?

Human: 30 000Roundworm: 19 000

Fruit fly: 13 500Arabidopsis: 25 000

Yeast: 6 300E. coli: 3 200

# of genes

DNA content is the same in each cell type

RNA profile is different in each cell type

Each cell uses (transcribes) only a small fraction (10-15%) of all the genes in the genome into RNA

How transcription works?

Transcription begun and mRNA elongation is underway

Transcriptase enzyme binds to the double stranded DNA, separates strands and the DNA 'eye' opens.

Elongation continues till the end of the transcription unit

RNA viruses (Retroviruses)

Viruses

The discovery of reverse transcriptase

Double stranded DNA viruses

Single stranded DNA viruses

• Do not contain DNA

• first recognized to cause certain cancers in animals

• Tumor cells have genetic-like stability

• RNA is not suitable for stable transfection

HOW RNA tumor viruses cause cancer???

The discovery of reverse transcriptase

Already known: certain bacterial viruses could integrate their DNA into their hosts' chromosomes and persist as stable genetic elements

Howard Temin proposed that RNA tumor viruses can cause permanent alterations to cells by integrating into host chromosomes.

RNAs first has to be converted into DNAs, which could then become integrated.

If all the above is true there MUST be a mechanism to convert RNA into DNA !

Is the Central dogma a “holy cow”?

?is there a way back???

Two separate research teams, one led by Temin and the other by

David Baltimore, simultaneously discovered the elusive RNA-copying

DNA polymerase in purified virions – after many years of painstaking

laboratory work.

The discovery of reverse transcriptase – the breaktrough

1970:

1975:

Temin and Baltimore shared the Nobel

Prize in physiology or medicine for their

discovery of reverse transcriptase.

Reverse transcriptase uses a single-stranded RNA template to create a double-stranded DNA.

What does reverse transcriptase do?

copy

c c c

How can it be used in research?

transcription

transcription

transcription

AAAAA

AAAAA

AAAAA

mRNA

mRNA

mRNA

• Excellent tool to study genes that are actually expressed in a certain type of cell/tissue/organ.

• Monitor gene expression changes due to environmental effects

Reverse transcription

cDNA analysis

Let’s start!

total RNA

tRNArRNAmRNA

~ 1%

• Most of the RNA is unimportant for us (tRNA, rRNA)

• mRNA population consists of about 3-5000 different kind

• Strong secondary structure – enzyme cannot work

AAAAAOnly mRNA has a poly-Adenin tail at the 3’ end

RNA isolation

RT reaction – step by step

AAAAA

AAAAA

AAAAA

65 ºC + ice

37 ºC

TTTTT

TTTTT

TTTTT

TTTTT

Reverse transcriptase

dATPdCTPdGTPdTTP

RNase inhibitor

mRNA

mRNA

1. denature

AAAAATTTTTmRNA

2. anneal + elongate

TTTTTcDNA AAAAA

37 ºC

RT

cDNART RT

ready

What shall we do with our cDNA now?

Problems:

• Vast variety of cDNA molecules (at least 3-5000 different kind)

• Only one copy DNA per mRNA strand exist

• Abundant (housekeeping) genes give 90% of the mRNA population

• They mask our gene of interest

Solution: we need a reaction that is

• able to amplify our low-abundance gene AND

• specific enough not to amplify other cDNAs

+++ Plus we need an enzyme that is able to copy DNA

The DNA replicates its information in a process called “replication”

DNA copying in a test tube

Can we use it on a cDNA template?

The enzyme who does the job:DNA-DNA polymerase

5` 3`

How DNA-DNA polymerase works

5`3` 5`

3`

5`Oligomer

- what if we denature and attach an oligo facing BACKWARDS…

1985: Kary B. Mullis

5`Pol

3`

5`3`

5`3`

5` oligomer

Pol

5` oligomer

We made one copy strand!

We made a second one!!

… and repeat it over and over again?

5` 3`

Pol

Polymerase Chain Reaction (PCR)

3 cycles – 2 4 cycles – 4 5 cycles – 8

..

10 cycles – 256..

20 cycles – 4 194 304..

30 cycles – 4 294 967 296

Cycle #4

Cycle #5

Kary B. Mullis: “what if we attach an oligo facing backwards?”

1993: Nobel Prize in Chemistry

1985:

Having good times - and inventing PCR

– an idea conceived while cruising the Pacific Coast Highway from San Francisco to Mendocino on a motorcycle.

How to amplify our gene of interest from the cDNA “soup”?

TTTTTcDNA AAAAA

TTTTTAAAAAcDNA TTTTT

AAAAAcDNA

PCR

Gel visualization

Gene #2

750 bp

500 bp

Gene #1

Gene-specific primers

RT–PCR at the bench

total RNA + oligodT

37 ºC – 1 hour

anneal + elongate

65ºC – 10 min

denature

Add:

Enzyme

dNTPs

RNasin RT ready

RT:

PCR:

DNA pol

dNTPs

primers

Buffer

MgCl2

95ºC3 min

denature amplify

95ºC – 30 sec55ºC – 30 sec72ºC – 1 min

72ºC10 min

finish

PCR ready

template

1-5 ul

Gel analysis

30 cycles

Applications of RT-PCR

• Cloning genes’ expressed forms (not genomic version)

• Monitor a gene’s expression level in any tissue

• Monitor expression changes following treatments

• Sophisticated RT-PCR: The real time PCR

• sequencing a whole mRNA profile

• EST (Expressed Sequence Tags) – database

• Microarray (DNA chip)

• Diagnose and easily differentiate between different cancer types

• Early detection of hidden illnesses

• etc…

Questions?

Resources, references

http://www.bookrags.com/sciences/genetics/reverse-transcriptase-gen-04.html

vcell.ndsu.nodak.edu/~christjo/vcell/animationSite/transcription/movie.htm

http://www.ambion.com/techlib/basics/rtpcr/index.html

….should you need more info just google around. RT-PCR is everywhere on the Web…

http://en.wikipedia.org/wiki/RT-PCR

Denaturation - separate parent strands in preparation new strand synthesis

Annealing - “stick” primers to the parent strands to prime DNA synthesis

Extension - addition of nucleotides, one at a time, to the growing end of the DNA strand (3’ end) using the parent strand as the template

PCR – A quick overview