Most genetic testing is carried out on deoxyribonucleic acid (DNA); mutations causing human disease can usually be found by DNA sequencing or other methods of analysis and DNA extracted from white blood cells is largely representative of that found in all the other cells of the body.
However, there are certain situations where analysis of RNA rather than DNA is preferable.
Before we describe one situation where it is preferable to analyse RNA than DNA (splicing), it is useful to review the central dogma of molecular biology.
The central dogma states that DNA codes for messenger ribonucleic acid (mRNA), which in turn codes for protein, the functional product of genes. After the mRNA has been made from the DNA template (transcription) it must be processed to remove the stuffer sequences (the introns) located between the exons, in a process called splicing.
This is a highly regulated process and relies on particular sequences of bases around the exon/intron boundaries, the splice sites.
Mutations occurring in splice site regions
Mutations are frequently identified which affect conserved sequences in splice site regions but it is hard to be certain what the exact effect will be on the splicing process. Possible outcomes are:
• Exon skipping where an exon is absent from the final mature mRNA. This could cause a frameshift effect, or just lead to exon exclusion.
• Inclusion of intronic sequence. This could cause a frameshift effect, or lead to additional amino acids present in the protein.
• Activation of a ‘cryptic splice site’, where the effect of the mutation is to change the sequence so that it mimics that of a naturally occurring splice site.
• No effect on splicing.
It is not possible to anticipate the effect of splice site mutations from the DNA sequence. In order to find out the effect of these mutations, and therefore how likely they are to cause disease, the RNA itself can be analysed.
RNA analysis
RNA analysis can use similar sequencing technologies to that used for DNA analysis, but there are several factors which make this a more taxing undertaking.
Some genes are active (‘expressed’) in most of our cells, so the RNA and protein products of these genes can be extracted from the blood, like DNA.
However, many genes are only expressed in particular tissues so in order to obtain RNA from these genes an alternative source may be needed (relatively easy from skin or saliva, less so from internal organs and the brain).
Furthermore, even if a suitable source of RNA is available, RNA is much less stable than DNA and is prone to being attacked by RNase enzymes, so speed and care are needed during sample handling and RNA extraction.
This inherent delicacy of RNA can be circumvented to some extent with the use of proprietary sample stabilisation products such as PAXgene™ blood collection tubes and RNALater™.
An additional problem with RNA analysis is that in some cases, if the mutation being sought introduces a ‘stop’ codon, a proof-reading system targets the RNA molecules for degradation in a process known as nonsense-mediated decay.
cDNA sequencing
In order to use the same techniques to analyse the sequence of RNA bases which are used for DNA analysis, the RNA is converted back to complementary DNA (cDNA) using a viral enzyme known as ‘reverse transcriptase’.
This reverse transcription reaction uses primers in a similar manner to a PCR reaction; primers can be gene-specific, so that only cDNA from a gene of interest is produced, or can be generic, either complementary to the poly-A tail of the mRNA molecule, or short random primers can be used which will bind in multiple places along the RNA molecules.
Once the cDNA has been made it is much more stable and can be used as a template for subsequent Sanger or next-generation sequencing assays. Sequence data can be aligned to a reference sequence and scientists will look for missing exons or intronic material, indicating that the normal splicing process has been disrupted.
Minigene assays
Minigene assays are a useful tool for looking for splicing changes when an RNA sample is unavailable, for instance when the gene of interest is not expressed in a tissue which is easily obtained, or if the patient is deceased.
DNA from the patient is extracted and PCR is used to amplify the gene of interest, including any mutations present in the patient’s sequence. This PCR product can then be cloned into a vector, a circular DNA molecule, and is transfected into cultured cells.
The vector includes the necessary signals needed for the cells to express the gene and the RNA can be extracted from the cell culture and reverse-transcribed and sequenced as if from the patient’s own tissue.
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