Question

1. What is meant by a “house-keeping” gene? Why is performing PCR on these an important control in qPCR?

2. Will the relative transcript levels of a gene always be the same as corresponding protein levels? Discuss why or why not.

3. What is meant by real-time PCR? In your answer, explain the terms cycle threshold and SYBR green.

Answer 1

House-keeping genes are also known as "Constitutive genes", as these genes are constitutively expressed in all cells in normal as well as diseased condition. The genes are transcribed at a constant level and are required for carrying out basic functions by the cell.

Now, these genes are an important control used while performing qPCR/ Quantitative PCR. This particular PCR assay measures the amplification or is for quantification. As house-keeping genes are constitutively transcribed at a constant level and are considered to be ideal can be best served as the control, when quantifying other gene products. This is also called Normalization whose purpose is to remove the non-biological variation as much as possible and as HK genes remain stable in normal as well as pathophysiological conditions, it serves best for the purpose.

Another question remains that whether the relative transcript of the gene will be the same as the protein level quantified.

Well in general the answer to this is No. As consideration for the same gene, theoretically, the mRNA content and protein should be the same, but practically it depends on various factors such as half-lives of mRNA and proteins.

We will see for two cases:

1. Constitutional/ house-keeping genes: It may be 'Yes' in the case of this type, but half-lives should be taken into consideration practically. e.g. actin, ubiquitin.

2. Tissue-specific genes: This is 'No' for the tissue-specific genes as they are expressed in response to some induction/repression. Some times, when the gene translation is not required, the transcript produced will be sequestered/blocked form translation by miRNAs or small RNAs which are regulatory in nature. e.g. insulin. The protein will be expressed when the cell senses the need for it otherwise the mRNA will be blocked even though it is synthesized. This is one of the ways you can look into it, but there are other factors such as post-transcriptional modification, half-lives, etc.

RT-PCR :  

Reverse Transcription Polymerase Chain Reaction which serves the purpose of quantification of specific RNA. As mostly PCR was designed to amplify DNA by the principle of unwinding and winding of double helix including replication, so that same DNA should be amplified, but what about RNA? So this modified version of PCR helps to convert RNA into DNA via reverse transcriptase enzyme and then amplification of DNA which is a specific fragment got from RNA. The DNA here formed is cDNA (complementary DNA) which is specific DNA amplification.

Cycle Threshold:

When qPCR experiment is performed, the amplification is usually measured in amplification plot, which is a sigmoid curve having three curves, first background curve in which there is a very low signal, second is exponential phase where the amplified product is higher and visible and last is the plateau phase, after which there is no increase in signal

The cycle threshold can also be termed as cycle no. or ct no. which represents the value that crosses the background fluorescence signal. When the reaction is going on the background fluorescence threshold is a low value, but the reaction goes on, there comes a point where the threshold value is more than the background value, this value is cycle threshold. At this point, the detectable no. of the amplicon is generated. This usually occurs during the exponential curve of the reaction.

SYBR green:

It is an asymmetrical cyanine due used in the visualisation of nuclei acid or used to stain nucleic acids during fluorescence or agarose gel electrophoresis reactions. It is used as an alternative to EtBr (Ethidium Bromide) widely used to stain DNA, due to its less hazardous property. It shows more affinity towards dsDNA than the ssDNA and RNA.

I hope this answer helps you!

Best Regards!