I currently aid in a research project based on Biofuels within a university. With a master’s degree in Biotechnology from Glasgow, I do have a strong educational background in Biology. Also, having worked as a Laboratory Assistant in an academic setting, I am quite familiar with several common laboratory techniques. During my spare time I work with a few charities and volunteer at my local hospital to assist them when their workload exceeds a comfortable level. I am also the head administrator for a non-profit organisation which works on the education of children in several poverty-ridden countries.
The effect of low calorie diets on people with Type II diabetes
In a study of people with Type II diabetes starting a low calorie diet it was found that the levels of the transcript for the gene UCP2 was significantly reduced (>3 fold), but the level of the protein product was found to be unchanged in the same cells. What techniques could be used to investigate the changes in the transcriptome and proteome in this type of study? Discuss the relative merits of these technologies and the implications of the lack of correlation between the transcript and protein levels in the example described above.
Insulin is an important peptide hormone produced in the pancreas of the human body. The main function of the hormone is to maintain blood glucose levels. All living tissue requires adequate glucose to function appropriately. Insulin converts excess glucose into glycogen and stores them within these tissues. Excess glucose within the bloodstream that cannot be converted to glycogen gets converted to fat. Hence, this hormone is essential in regulating both carbohydrate and fat metabolism. Diabetes mellitus 2 (DM2) is a chronic disease in which insulin levels are affected. This disease is categorised as adult onset and could be triggered by various factors (1). Insulin may not be adequately secreted in patients diagnosed with DM2 or in some cases; tissues may have become insulin resistant. Insulin resistance occurs when receptors within the tissue become resistant to insulin, thereby not absorbing glucose for its functioning. In both cases, the glucose levels in the blood are abnormally high and have to be treated. Unlike diabetes mellitus 1, patients diagnosed with DM2 are not insulin dependent and only 40% of them require external insulin (11).
Recent studies show that inhibition of uncoupling protein 2 (UCP2) protein expression in DM2 patients improved insulin secretion and action drastically (2). It was also studied that the transcript levels of the UCP2 gene in patients who followed a low calorie diet were reduced nearly threefold. However, the protein expression levels remained the same despite the reduction in transcript levels. For expression of the protein, UCP2 genes are first transcribed to their mRNA, which then gets translated to protein. Transcripts are products of transcription of the gene and they contain both intron and exon sequences. These transcripts are then spliced off the introns sequences and capped to obtain the mRNA to which a poly nucleotide tail is added. With the help of transcriptomics and proteomics, an in depth knowledge of the transcriptome (set of transcripts) and proteome (level of protein expression) can be obtained.
mRNA transcripts of the UCP2 gene can be studied using various transcriptomics tools. The most effective and reliable of these tools include microarray analysis, high throughput sequencing methods and RT polymerase chain reaction.
Microarrays can be used to study how low calorie diet affects the level of transcripts of the UCP2 gene. mRNA of the gene is first reverse transcribed to their cDNAs. These cDNAs can be obtained from various cDNA libraries hence making the process efficient and less time consuming. The cDNAs (probe) are then attached to a glass or nylon membrane in the form of an array. The array membrane is usually hydrophobic to prevent hydrolysis of cDNA molecules as well as to increase the adhesion of the probe to the membrane (10). Transcript samples are then obtained from the patient both before and after low calorie diets. These samples are labeled with fluorescent or radioactive labels and are then made to hybridise to the array containing the probe. The amount of fluorescence or radioactivity can be recorded and this data correlates with transcript levels. Procedures involving microarrays are fairly simple, feasible and it does have a high capacity for multiplexing as well. It is of advantage in this study as the sequence of the UCP2 gene is known, which allows easy probe production.
Although microarrays are preferred in this study, high throughput sequencing methods are widely used as well to study the transcriptome of a gene. Sequencing is advantageous as it can detect transcript levels when the sequence of the gene is unknown. This, however, is not possible in microarrays as the production of the probe requires the sequence of the gene. Sequencing can also avoid errors in hybridisation and detect the presence of single nucleotide polymorphisms (SNPs) in the sample transcripts (7). These samples are first reverse transcribed to their cDNA before sequencing methods. The two most widely used methods in analysing transcriptome by sequencing are Solexa/ Illumina and ABSOLiD. Amplification before sequencing is carried out by bridge PCR in Solexa and emulsion PCR in ABSOLiD. Both of these methods result in clonal amplification of the sample template.
Sequencing in Solexa is done by synthesis. Here, each of the four nucleotides is labeled with different fluorescence labels along with a blocker. In every cycle, all four nucleotides are added simultaneously and the color of the fluorescence defines the correct nucleotide. For every subsequent cycle, the blocker and label are washed away. ABSOLiD, on the other hand, uses sequencing by ligation where labeled dinucleotides or oligonucleotides are used. In case of dinucleotides, a set of four dinucleotides are labeled by a single marker. Sequence in every position is determined by two or more successful ligations. Solexa sequencing can run sequence lengths of around 100bp whereas ABSOLiD can read lengths of around 50-75bp (7). In both these methods, the level of fluorescence obtained after sequencing the sample cDNA correlate with the total amount of transcripts present. In this study of UCP2 gene, sequencing methods are useful to detect post transcriptional mutations.
Apart from the above mentioned techniques, polymerase chain reaction can be used to obtain highly precise transcript levels of a sample. Duplex real time quantitative reverse transcriptase PCR is a highly sensitive reaction to quantify mRNA of a sample (5). It detects and measures product level generated in every cycle of the reaction. Sample transcripts are first reverse transcribed to their cDNAs. A fluorescent labeled probe is then hybridised within the sample cDNA sequence. During PCR process, primers move towards the probe. This probe eventually gets degraded to emit fluorescence which are detected to estimate transcript level of the sample (10). Real time qRT PCR can be used in both absolute and relative quantification of gene expression. Although transcriptomics helps us study the expression of genes and transcript levels, the results need not correlate to the protein expression.
Proteomics is the study of all proteins expressed within a gene. A better understanding of UCP2 protein expression in DM2 patients can be obtained using various proteomic tools. Separation of the protein is the first step to study their expression. For this purpose, various chromatographic and electrophoretic methods are often carried out as primary step before proceeding with protein identification by mass spectrometry.
Two-dimensional difference gel electrophoresis is an improved version of 2-D gel electrophoresis. Unlike the traditional method, error due to gel variation is eliminated in 2D-DIGE (10). This method is used to separate as well as detect protein expression within the samples. 2D-DIGE has the ability to detect minute differences in the protein expression and hence is highly sensitive. Protein samples are first labeled with fluorescent dye and are then separated on the basis of their isoelectric point and molecular mass. Two dyes are used in this method, the control samples are labeled with Cy3 fluorescent dye and the test samples are labeled with Cy5 fluorescent dye. The normalisation of fluorescence intensity eliminates gel to gel variation. Staining methods are replaced with laser scanners in 2D-DIGE which make them more sensitive and less time consuming. Protein spots obtained in the 2D-DIGE are then excised and digested with protease enzyme. This enzyme induces proteolysis and breaks the protein down into peptides. These peptides are then run through mass spectrometry for identification of the protein.
Stable Isotype Labeling with Amino acids of Cells (SILAC) is a proteomic tool used to quantify protein expression and also used to study intra protein interactions (4). In this study, it can provide accurate protein expression comparisons in samples obtained from patients both before and after following a low calorie diet. One sample set is cultured with labeled lysine and arginine amino acids whereas the second set is cultured with the heavy isotopes of these amino acids. The arginine and lysine containing peptides of the second sample set are heavier due to heavy isotope labeling (3). This difference in their masses enables mass spectrometry to differentiate even identical peptides. Measurements in SILAC are made at the peptide level and hence have very high sensitivity.
Mass spectrometry is the primary tool for quantitative and qualitative analysis of a protein. Mass of different peptides within a protein can be measured using this technique (14). Protein samples are first fragmented to their peptides by addition of trypsin. These peptides are then fed into the mass spectrometer in which four of the following steps are carried out; Ionization, Acceleration, Deflection and Detection (8).
The peptides are first converted to positive ions by bombarding them with an electron beam. This removes electrons within the peptide molecule, making them positively charged ions. These ions are then accelerated and passed through a magnetic field. While passing through this field, ions get deflected based on their mass and charge. The lighter or more charged the ion is, the more they get deflected from their path. These factors together form the mass/charge ratio (m/z). This value is obtained in the detector in the form of a mass spectrum. This result is then compared to other mass spectra in various databases. Commonly used databases include SwissProt, Protein identification database (PRIDE) etc. Improved mass spectrometry methods used currently include Matrix Assisted Laser Desorption Ionization- Time Of Flight- Mass Spectrometry (MALDI-TOF-MS) and Liquid Chromatography- Electrospray Ionization Tandem- Mass Spectrometry (LC-ESI-MS/MS) (13).
Once analysis results using transcriptomics and proteomic tools have been obtained, the UCP2 gene and protein expression can be better understood. However, tools for quantifying protein abundance have seen improvement in technology only over recent years (9). Researchers used to try and correlate the data obtained from transcriptome analysis to obtain protein expression levels. As mentioned earlier, the protein expression after DM 2 patients underwent a low calorie diet, remain unchanged. The transcript levels however, reduced threefold. This lack of correlation between the transcriptome and proteome could be due to post-transcriptional modifications and/ or due to errors in the transcriptomics/ proteomic analysis (6).
Post transcriptional modifications to the transcript include splicing of the introns sequences, capping and addition of a poly nucleotide tail. The length of this nucleotide tail determines the efficiency of the translation process. Cis-regulators or RNA-binding proteins may bind to the mRNA as well and this either induces or suppresses protein translation (16). In some cases, a protein itself may influence mRNA down-regulation thereby lowering transcript levels. Improper correlation between the transcriptome and the proteome may be attributed to the above mentioned reasons. Studies also show that if variability of transcript expression levels throughout cell cycle is low, it may not have much correlation with protein expression levels (15).
Although methods used to study UCP2 transcript and protein expression can be done using various transcriptomics and proteomics tools, the exact understanding of the proteome and transcriptome correlations require further analysis. Inhibition of the UCP2 protein has shown to improve insulin secretion and action in diabetes mellitus 2 patients. Studies also show that treatment with UCP2/AS oligonucleotides could obtain an 85% reduction in UCP2 protein expression levels (2). UCP2 is therefore a potential target for further studies related to therapeutics of Diabetes Mellitus 2.
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