Editor’s note: Cosmetics & Toiletries magazine wishes to acknowledge that this article by Remona Gopaul is the first based on a capstone project for the University of Cincinnati’s Masters in Cosmetic Science program, led by R. Randall Wickett, PhD. Cosmetics & Toiletries anticipates future articles from students of this program.
Deoxyribonucleic acid (DNA) contains the genetic information needed for the development and functioning of all living organisms,1 and as most elementary level science students will know, the segments of DNA that carry this genetic information are known as genes. Information can travel from DNA to messenger ribonucleic acid (mRNA) through a process called transcription, from mRNA to complementary DNA (cDNA) via reverse transcription, and from mRNA to protein via translation, as illustrated in Figure 1.1 By identifying which mRNAs are present in a cell, one can understand which genes are active in that specific cell type.
With the completion of the Human Genome Project and advancement of genomic technologies, cosmetic companies have begun to utilize genomic techniques to understand the expression of genes and their relationship to particular skin attributes, as well as to test topical ingredients and formulations.
Genomic testing can be conducted on a small scale via techniques that measure the expression of single genes or small groups of genes, or on a global scale via methods that measure the expression of thousands of genes simultaneously in one experiment.2 A combination of different techniques is the best approach to investigate the gene expression profile of skin under different conditions.
Small Scale Techniques
Northern blot: Historically, northern blot is one of the most standardized methods for detecting and quantifying RNA sequences. The first steps involve isolation of RNA and fractionation by gel electrophoresis. The RNA is then blotted onto a positively charged nylon membrane and hybridized with a labeled cDNA probe specific to the gene of interest. The intensity of hybridization, i.e. the process of joining/bonding (covalently) two complementary strands of DNA, is then detected by x-ray film and can be quantified by densitometry.3 Although the northern blot method is well-established and relatively simple to perform, the processing time is consuming and this method is not as sensitive as most modern techniques. In addition, radioactivity often is used. It is important to note that the time it takes to conduct a northern blot experiment depends upon how long the RNA is incubated with the test material, thus the process requires preparation work such as making gels, which other methods do not require. Due to these limitations, this method is not often the method of choice for gene expression and is consequently being replaced by more modern techniques.
RT-PCR: Reverse transcriptionpolymerase chain reaction (RT-PCR) amplifies cDNA made for a specific mRNA transcript. The first step involved in a RT-PCR experiment is incubation of the application with a specific tissue type. RNA is isolated from the tissue at the end of the incubation period followed by the production of cDNA through the process of reverse transcription. Each copy of cDNA is amplified by PCR. The most common method for conducting a RT-PCR experiment is real-time PCR, which involves continuous monitoring of the double-stranded DNA in real time rather than at the end, as with conventional PCR.2
RT-PCR, especially real-time PCR, is considered one of the most sensitive techniques for gene expression and is highly quantitative and reproducible. This method can also be expensive since it only analyzes a few hundred genes at a time and can pick up RNA carryover that may lead to false positives. Nevertheless, real-time PCR is emerging as one of the best techniques used for gene expression. There are two types of real-time PCR: the SYBR green method, which uses dyes that fluoresce when bound to double-stranded DNA; and the TaqMan method, where DNA-based fluorescent probes are used to measure the accumulation of fluorescent signals against a preset threshold.
TaqMan real-time RT-PCR is the method most widely used in the cosmetics industry because it gives more comprehensive quantitative data than other techniques. Quantification of mRNA transcripts from TaqMan real-time RT-PCR is obtained by comparing transcript levels with those from “housekeeping genes” such as glyceraldehyde phosphate dehydrogenase and cyclophillin. These genes are considered to be invariants and are often used as control genes for gene expression experiments.2
The data from the TaqMan method reports a cycle threshold (Ct) value, which is defined as the number of cycles required for the fluorescent signal to cross the predefined threshold. Ct levels are inversely proportional to the amount of target gene.4 Standard real-time reactions undergo 40 cycles of amplification. Ct values below 20 indicate strong positive reactions and an abundance of the targeted DNA; Ct values of 30–37 are positive reactions indicative of moderate amounts of target DNA; finally, Ct values of 38–40 are weak reactions indicative of minimal amounts of target DNA.4
Global Scale Techniques Microarrays: Microarrays are constructed by spotting cDNA or oligo- nucleotides of known gene transcripts in a gridded pattern on a glass slide or chip. There are two main microarray platforms, slide-based arrays and oligonucleotide arrays. Although extremely comprehensive, microarray methodologies only provide information about the expression and condition of specific genes at a particular time, which may be a limiting factor based on study objectives.
In a slide-based microarray experiment, the material being investigated is first incubated with a specific tissue type such as human equivalent skin cultures, individual skin cells such as fibroblasts, or skin biopsy samples. RNA is isolated from the tissue at the end of a predetermined incubation period. Reverse transcriptase is then used to transcribe the mRNA into cDNA. The nucleotides used to synthesize the cDNA are labeled with either a green or red dye, one color for reference conditions and the other for experimental conditions. The slide is incubated overnight with both reference and experimental cDNAs to allow hybridization, after which the slides are washed to remove any unbound cDNAs. Subsequently, computerized images are produced by scanning to detect the grid spots containing cDNAs labeled with green dye, red-labeled cDNAs, and a combination of the two colors (yellow), which represents transcripts that are expressed under both sets of conditions.2, 5, 6
Oligonucleotide arrays are based on similar principles as the glass slide method; however, rather than assessing the activities of two samples in each experiment, oligonucleotide arrays measure the activity of one sample in each experiment. The main difference in the experimental protocol for oligonucleotide arrays is that cDNA is allowed to go through in vitro transcription back to RNA (cRNA). This cRNA is labeled with biotin, fragmented and added to the array for hybridization. The array is washed to remove any un-hybridized RNA. Finally, the entire array is scanned with a laser and the information is stored electronically for quantitative analysis.5,7
Oligonucleotide arrays often utilize full genome chips including transcripts from an entire organisma. These allow for the measurement of all gene expressions in one experiment—an advantage over the glass slide method, where the expression of only a specific gene is measured. Although oligonucleotide arrays, being of a larger scale and more technologically advanced, are significantly more expensive than the glass slide method, they also produce fewer errors since only one sample is tested per experiment, preventing cross hybridization.2
SAGE: Serial Analysis for Gene Expression (SAGE) is a genome-wide technique that identifies and quantifies new gene transcripts rather than relying on pre-existing information about a specific sequence.8 SAGE measures a short nucleotide sequence (tag), of a particular transcript located on the 3' end of each cDNA. The number of short sequences is proportional to the amount of mRNA in the original sample; therefore, the quantification and identification of specific gene transcripts can be obtained with this technique.8 SAGE can be useful in the discovery of new biomarkers for skin disease and aging since it does not require prior sequencing knowledge of gene transcripts like other gene expression techniques. Although SAGE is a novel approach to understanding gene expression, the technique can be time-consuming as the protocol is rather long and requires the individual conducting the experiment to perform much preparation and analysis work, unlike other simpler techniques such as microarray.
RNA sequencing: Similarly to SAGE, RNA sequencing is a genome-wide technique that generates the sequences for the RNA in a specific sample. The first step in RNA sequencing experiments, as with most gene expression techniques, is to isolate mRNA from the pre-incubated tissue. cDNA fragments are created via reverse transcription. Sequencing adapters are then added to each cDNA fragment and a short sequence is obtained from each cDNA using high-throughput sequencing technology.9, 10 The resulting sequences are then aligned with the reference genome.
This assay provides a sensitive, digital count of all expressed mRNA in the sample. Since the signals are based on DNA sequencing and not hybridization as with microarray, false-positives are few, making the method statistically better than most other large scale techniques.10 As with microarrays, RNA sequencing produces large data sets, requiring specialized expertise and complex, expensive software programs to analyze and interpret results. RNA sequencing may be a possible replacement for microarray analyses in the future, and it may revolutionize the way gene expression experiments are conducted.
Microarray for Claim Substantiation
As noted, the most common gene expression technique used in the personal care industry is the microarray method. This method is intended to give a global gene expression profile of a specific application. Global gene expression profiles often result in large data sets consisting of thousands of genes and to date, not many skin-related application programs and/or experts have been established to analyze such large amounts of data. So although having large data sets in an experiment may be welcomed, not having the programs or bioinformatics to analyze them can make them overwhelming.
Unfortunately, this limitation has not kept some raw material vendors and cosmetic companies from using such methods as microarray to substantiate efficacy and claims. By using only global gene expression data, companies do what is known as fishing for genes in order to substantiate efficacy. In this scenario, researchers scan the data sets from microarray experiments and select specific genes of interest to highlight without fully understanding their function in relation to skin. For example, to measure the genetic effects of an antiaging compound, one may conduct a microarray experiment in which the results show thousands of genes (based on statistical parameters) being up-regulated or down-regulated. But since little research exists on skin genes, software programs designed to analyze gene expression data from microarrays are not of much use. Hence, the researcher would need to conduct in-depth literature reviews on all of the genes to understand the function and relation of them to skin.
Once this information is retrieved, the researcher would then fish for genes to substantiate a particular cosmetic claim. If the claim is “helps to prevent lines and wrinkles” and the data set includes the up-regulation of genes such as collagen and elastin—genes known to be positively associated with young skin, the researcher would choose these genes as substantiation for that particular claim. This fishing process is how researchers narrow data down from thousands of genes to small groups of single genes. Companies also have been generating pathways from these large data sets and using the fishing process to chose specific genes shown to be associated with a particular skin biological pathway.
A more effective approach of using microarray data sets to validate claims would be to confirm genes selected through the fishing process with a smallscale methodology such as RT-PCR. The genes from these data sets should then be confirmed using a proteomic technique to analyze the correlation between the mRNA levels and protein levels. Once the proteins are confirmed, conclusions can be made on the effects of a cosmetic technology on gene expression and subsequent protein synthesis.
Genomics in Product Development
In addition to efficacy screening and claims substantiation, companies have been using genomic screening to test raw materials and formulations for toxicity. Known groups of genes associated with sensitive skin and/or irritation can be used to gain an understanding of the toxicity potential of specific materials. Genomic techniques also elucidate the effects of a raw material or final formula on various biological pathways in the skin; by using a method such as microarray, the investigator can obtain a signature profile of the gene activity of a particular topical application. From this, genes can be categorized by biological function or pathways to give an overall assessment of the effect of the application on gene expression. For genes of interest, a validation technique such as RT-PCR should be used to confirm their expression.
Along with their use in formulation and product development, genomic techniques provide scientists with an understanding of the biological processes and genes involved in different skin types, such as young and old skin. In addition, by obtaining global gene expression profiles for skin of different conditions and ages, scientists will further the understanding of the skin aging process.
Study Design Considerations
Prior to conducting a genomic experiment on a topical product or ingredient, the investigator should determine the objectives of the study and address relevant questions such as:
1. Is the objective of the study to understand the regulation of a particular gene, or to obtain a global gene expression profile of the topical application on skin?
If the regulation of a particular gene is being investigated such as collagen type I alpha (COL1A) or elastin, then a single gene technique such as northern blot can be used. If groups of genes are being investigated such as genes associated with keratinocyte differentiation, then TaqMan RT-PCR should be used, where the manufacturer of the PCR cards can design a card containing all the primers for the gene group, subsequently allowing for the simultaneous measurement of gene expressions for the entire group in real time.
If a global gene expression profile is being investigated, then a high throughput methodology such as oligonucleotide microarray should be used, where all the transcripts from the desired genome are represented on one DNA chip. As noted, using whole genome chips allows the investigator to measure the expression of all of the genes from the genome in one experiment, providing a comprehensive gene expression profile of the application.
2. Is the experiment intended to discover new mechanisms of action for the topical application, or to confirm specific claims?
If the objective of the experiment is to discover new mechanisms of action of an application, then a high throughput technique such as microarray or SAGE should be used, where thousands of genes can be investigated simultaneously. The data can then be categorized by biological function or pathways to potentially reveal new information of the application on gene expression. If the data indicates novel discoveries, the investigator may want to confirm the expression of specific genes using a small scale technique such as TaqMan RT-PCR. If the objectives of the experiment are to confirm or substantiate specific claims, a small scale technique such as TaqMan RT-PCR should be used to measure the expression of the targeted genes relevant for claims substantiation. 3. Is the sample and/or tissue type relevant? Skin biopsy samples often are preferred when conducting skin genomic experiments since the data will represent the activity of the application on real skin tissue. However, for low-budget and screening experiments, human equivalent skin tissues are frequently used. In addition, if the objective of the experiment is to measure the effect of the application on a particular cell type, then individual skin cells such as fibroblasts or keratinocytes can be used.
The future will undoubtedly introduce improved techniques, experimental designs, bioinformatic tools and novel approaches for skin genomics. Innovations in this area may allow the discovery of new biochemical and genetic pathways in the skin, and new regulators of skin diseases and skin aging, as well as new mechanisms of action of novel ingredients and products. This is an exciting time for cosmetic scientists and formulators who are looking for new stories and validation techniques to test and substantiate ingredients and final formulations. However, these techniques, as with all new approaches, should be studied and reviewed extensively before being established as industry standards.
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