Molecular Genetics Lecture 9 pt. 2 - Detection and quantification of mRNA expression

Reverse transcription PCR, Microarray analysis, RNA Sequencing, Gene expression atlas

What are the steps of reverse transcription PCR?

1. isolate mRNA from different tissues/organs

2. reverse transcribe mRNA into cDNA

3. use primers specific to gene of interest to PCR amplify exons

4. gene expression is measured based on the amount of PCR amplified DNA molecules (End point PCR)

what is used in Real-time RT-PCR?

SYBR Green dye and TaqMan chemistry

What is Real-Time PCR used for?

quantitate the amount of specific amplified PCR product in each cycle in real-time to quantify the amount of cDNA

what are the steps of Real-time reverse transcription PCR?

1. add DNA intercalating dye SYBR Green in PCR which binds to double stranded DNA and emits fluorescence

2. RT-PCR machine equipped with a detector that quantifies PCR amplified DNA amounts based on flurescence intensity in each cycle

what are the 3 phases of PCR based on the amount of amplified fragment?

exponential, linear, plateau

what does Ct (cycle threshold) represent?

fluorescence level above background and detectable

what does Ct value measure?

how many PCR cycles required for the sample fluorescence to reach the threshold level

what is a low Ct?

high cDNA template amount, high mRNA expression

what is a high Ct?

low cDNA template amount, low mRNA expression

what does microarray analysis allow?

monitoring global gene expression changes in thousands of genes in an organism at the same time

what does microarray analysis require?

completely sequenced reference genome with annotated genes (exons) and a custom-made gene chip for each organism

in microarray analysis, what is used as a probe?

short oligonucleotide fragments of known exon sequences of genes

what is microarray analysis used for?

to detect differential mRNA expression in normal and cancer cells

what are the steps of microarray analysis?

1. develop a microarray/chip representing exons of all the genes in the genome

2. extract mRNA from normal and cancer cells

3. convert mRNA to cDNA and label with fluorescent dyes

4. hybridize cDNA to the same microarray

5. detect hybridization and data analysis

what color and dye are normal cells in microarray analysis?

CY3 green

what color and dye are cancer cells in microarray analysis?

CY5 red

if a gene is black in microarray analysis what does this mean?

no gene expression in normal and cancer cells

if a gene is yellow in microarray analysis what does this mean?

it is expressed equally in normal and cancer cells and there is no change in expression

if a gene is green in microarray analysis what does this mean?

it is expressed and upregulated in normal cells and downregulated in cancer cells

if a gene is red in microarray analysis what does this mean?

it is upregulated and expressed only in cancer cells and downregulated in normal cells

are gene chips with probes required for RNA sequencing?

no

what are the steps of RNA sequencing?

1. isolate mRNA

2. fragment and create a library

3. convert to cDNA

4. sequence by Next generation Sequencing technology (Illumina)

5. map the reads to the genes

how is gene expression quantitifed in RNA sequencing?

based on number of sequenced reads match to the gene (RPKM, TPM)

what does RPKM stand for?

Reads Per Kilobase of transcript per Million mapped

what does TPM stand for?

Transcripts per million mapped reads

in addition to mRNA, what can RNA sequencing also discover?

other functional RNAs like small RNA, microRNA, non-coding RNA

what are the pros and cons of RNA sequencing?

cheap and fast, but data analysis is complicated in organisms without a completed genome

what are the steps to creating a Human whole genome gene expression atlas

1. purify mRNA from various tissues (lungs, colon, eyes, brain, etc)

2. convert it to cDNA

3. perform Microarray analysis or sequence the cDNA by RNA sequencing

4. create a database to show mRNA expression of thousands of genes in each cell/tissue/organs of an organism