Gene expression: Synopsis, comparison of techniques and perspectives

Question 1

Name a primer problem that you have to handle when interpreting qPCR results.

Answer 1

1. Similar homologues and alternative splice products may be amplified by the same primers as the ones used for the desired amplicon.
2. Primer dimers.

Question 2

Can you extrapolate the protein expression from a cDNA analysis (RT-qPCR)?

Answer 2

No. Translational efficiency is not taken into account.

Question 3

What are biological- and technical replicates?

Answer 3

Biological replicates: Different samples.

Technical replicates: Same sample in different reactions.

Question 4

What three cateogries of errors are there in RT-qPCR reactions?

Answer 4

1. Technical variations between PCR reactions.
2. Technical variations between RNA/cDNA samples.
3. Biological variation between RNA/cDNA samples (genetic, epigenetic)

Question 5

When chosing a reference gene for your RT-qPCR, in what sense do you have to take the Ct-value into account?

Answer 5

Depending on the gene’s endogenous expression, it may be hard to interpret the result. High endogenous expression leads to a low ct-value, low endogenous expression leads to a high ct-value.

Question 6

When quantifying transcripts, what mehtods can you choose from? (3 qualitative methods, 2 quantitative methods)

Answer 6

Qualitative
1. Northern blot (gives mRNA size)
2. In situ hybridisation (gives spatial distribution in cells)
3. Microarray (gives a global overview of known genes)

Quantitative
1. RT-qPCR (gives specific transcript expression)
2. RNAseq (gives an unbiased overbiew, mRNA size and gene structure).

Question 7

Explain the concept behind picoliter limit dilution (digital) PCR.

Answer 7

1. The template is very diluted.
2. PCR reactions are ran in emulsions that traverse a temperature plate (denat, anneal, elong…)
3. Quantify the number of emulsions where PCR amplification takes place.

Question 8

What are the benefits of the picoliter dilution (digital) PCR?

Answer 8

1. It’s insensitive to PCR inhibitors (proteins, lipids)
2. It’s insensitive to primer dimers.

Question 9

What’s the benefit of running high-throughput PCRs?

Answer 9

There’s up to 10000 parallel reactions where the template is amplified by lots of different primer pairs.