Gene Expression Unit: Use sequencing to unveil a gene linked to obesity

What is the Ct value for the LEP obese sample and LEP lean sample, respectively? (Amplification plots)

22 and 22 -PCR product is detectable in cycle 22 in both LEP samples

Let us refresh our knowledge about the structure of an mRNA molecule. Which is the correct order?

5' UTR, Exon 1, Exon 2, Exon 3, 3' UTR, AAAA

What are the possible applications of the NGS technique?

All of these applications -Detecting generic aberration -Perform single nucleotide polymorphism (SNP) profiling -Perform gene expression profiling

Why do we need to reverse transcribe the mRNA to cDNA before PCR and sequencing?

Because the RNA cannot be used by the Taq polymerase

If genomic DNA left in your sample has been amplified as well, which melting curve would you expect to see?

C -See additional peaks all over the place bc it amplified the intron in the gDNA -Gives bigger PCR product = melts @ higher temp

Which value describes the sequence quality?

D (BAAAGECEE<EEDFEDF3DBDBB=A+==>9>>88?)

If the efficiency of the PCR is 100%, how has the quantity of product changed at the end of each cycle?

Doubled

Now that we have run the PCR and accumulated enough modified DNA for the NGS experiment, what is the next step?

Generate Clusters

Which of the 2 genes: Leptin (LEP) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) illustrates differential expression?

LEP -We see more reads from obese (blue) compared to lean pig (red) -LEP is good candidate gene for obesity

Now we know that there is 16 times more cDNA in the lean sample compared to the obese sample. However, for both LEP samples, the Ct values are the same. If you combine these facts, what can be concluded about LEP expression?

LEP expression is 16 times higher in obese compared to lean samples -Even if Ct value for LEP is identical in the 2 samples, after normalization it shows LEP is up-regulated in obese

How do we see that a gene is highly expressed?

Many reads will map to that gene

Before publishing this astonishment result it should be confirmed by another method. Which other method can be used to measure gene expression?

Quantitative PCR (qPCR)

What is the most important feature that a reference gene should have?

Stable expression in all samples

A GAPDH Ct of 24 for the obese samples and 20 for the lean samples indicates that...

The amount of GAPDH cDNA is lower in the obese samples compared to the lean samples - An inc in Ct value --> More amplification cycles needed to detect same amount of fluorescence vs other sample - Shows that initial sample of cDNA was lower in obese

In an mRNA sample some transcripts are highly abundant while others are rare. What would be the status of the corresponding cDNA sample?

The cDNA quantity will represent the mRNA quantity

What is the reason for different reference gene Ct/cDNA amounts in the two samples?

The efficiency of cDNA synthesis caried between samples -Efficiency of cDNA is influenced by remaining contaminants @ RNA samples (lipids,etc) -Reference gene usually equally expressed in all samples

When would we expect to see the negative control including water and qPCR mix to produce a signal?

The qPCR master mix is contaminated with the template

The Ct difference (delta G) between the obese and lean samples for the reference gene (GAPDH) is 4 cycles. What does this mean?

There is 16 times more cDNA in lean compared to obese samples -Difference of 4 cycles btw samples for GAPDH --> 2^4 = 16 times more cDNA in lean vs obese

Every read is mapped to the reference genome by alignment. Why?

To identify from which gene the read comes from

After excluding sequences with low quality score, what should we do?

Trim out the adapters