From Impossible to possible
The genetics of cancer has progressed from the impossible to a multifaceted mountain of possible. Comprehensive projects in whole genome sequencing and tumor genome sequencing are providing data to unravel the genetic predispositions of cancer.
The other half of possible lies in quantitative PCR. This platform technology can identify types of cancer, effective therapeutics, and aggressiveness of the disease from biopsy samples. Several leading diagnostics companies, including Genomic Health (Redwood City, CA), Myriad (Salt Lake City, UT), GenomeDx (San Diego, CA), Holigic (San Diego, CA), and Biotheranostics (San Diego, CA) use variations of quantitative PCR.
In this issue, we’ll take a closer look at how gene expression is used to understand disease and develop better diagnostics.
MRNA is the intermediary
To develop diagnostics, scientists need to understand how gene expression influences protein expression—or how often a gene is translated into a protein.
In some cases, the actual sequence of a particular gene does not differ between healthy and diseased—it is simply expressed more (or less) often in the diseased population. In other cases, there may be a combination of gene mutation and expression. In fact, it is important to look not just at the expression of one gene, but at the ratio of expression between two different genes.
Messenger RNA (mRNA) is the intermediary between genes and proteins: a gene is transcribed into mRNA and that mRNA is translated into chains of amino acids—the building blocks of proteins. It is almost impossible to isolate the total amount of a particular protein made by a cell, whereas techniques for isolating total mRNA content are in common practice, making mRNA the perfect intermediary to determine gene expression.
EASILY CONFUSED: Gene Expression vs. Protein Expression
Gene expression occurs before protein expression. It is when the information from a gene is used to make messenger RNA (mRNA). Genomics scientists measure the presence and abundance of mRNA in cells and tissues after gene expression.
Protein expression occurs after gene expression. It is the process after the gene has been transcribed into mRNA. The mRNA is then translated into amino acids that join together to make polypeptide chains. These chains fold to make proteins. Proteomics scientists measure the presence and abundance of proteins in cells or tissues after protein expression.
the beauty of hacking into mrna
One way to measure gene expression associated with disease is to determine how much of a specific mRNA is present amongst the total cellular mRNA. This can be accomplished using a technique called quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).
The first step in qRT-PCR is converting the isolated mRNA back into DNA. This is necessary because mRNA is highly unstable and it will not hang around long enough to be analyzed. This is accomplished by mixing the isolated mRNAs with an enzyme called reverse transcriptase (RT) and adding adenine, cytosine, guanine, and thymine (the nucleotide building blocks of DNA). mRNA is copied back into DNA when the DNA building blocks find their respective matches along the single-stranded mRNA sequence—same genetic information, but in the much more stable format of DNA. These pieces of DNA are called cDNA because they are copies of mRNA transcripts. For every mRNA present in a sample there will be exactly one cDNA produced. If we determine how many copies of a particular type of cDNA are present, we will know how much of the corresponding mRNA was made by the patient’s cells—and a diagnosis can be made.
To quantify the amount of a particular cDNA, a machine called a thermocycler combined with the process of polymerase chain reaction (PCR) is used. PCR is a way of making many copies of one specific DNA sequence. The final number of copies of that DNA sequence is in proportion to the beginning number. By measuring the number of new DNA strands created from a specific cDNA, it is possible to back-calculate and determine the initial amount of that cDNA—thus the initial amount of mRNA present.
Quantitative PCR enables measurement of the amount of a particular DNA produced by releasing fluorescent signals as new strands of DNA are created. The more fluorescence produced, the more copies of the cDNA being measured are present. The technique is also called Real-Time PCR because the fluorescent signal is measured in real time—as the reaction is happening. If we wait until the end to measure, it will be completely saturated and not yield good data.
COCKTAIL FODDER: WHERE DOES RT COME FROM?
The enzyme RT—which is used as the first step in quantitative PCR to convert RNA to DNA—was discovered in viruses called retroviruses, with HIV being the most well-known example. Retrovirus literally means “reverse virus” because they copy their genetic material (typically RNA) into DNA in order to incorporate itself into the host cell genome, working in a backwards cycle to assimilate into their target.