Mechanisms in expression microarrays, and their advantages and disadvantages.

Gene expression analysis is an essential aspect of molecular biology that enables researchers to investigate the transcriptional changes occurring in a cell or tissue under various conditions. Two commonly used techniques for gene expression analysis are expression microarrays and RNA sequencing (RNA-seq). Both methods involve measuring the abundance of transcripts from thousands of genes, but they differ in their mechanisms and have distinct advantages and disadvantages.

What are microarrays?

microarrays are a powerful tool for analyzing gene expression levels in living organisms. They are composed of thousands of individual DNA fragments that are immobilized on a solid support such as glass. By hybridizing these fragments with complementary target sequences in the organism of interest, researchers can gain insights into the expression patterns of specific genes.

Fluorescent reporter molecule is used to detect the hybridization. to detect specific amplicons Microarray detection is typically combined with PCR. in Microarray techniques use many different “probe”  to detect simultaneous, between different organisms, or differences between organisms of the same species. Microarrays are one of the powerful processes to characterize and detect of microbial pathogens with a high degree of specificity, but are less sensitive and expensive, so they are unlikely to find routine use for drinking water analysis in the future (Microarray – an Overview | ScienceDirect Topics, n.d.).

Microarray hybridization technology is a significant advance for differentiating Campylobacter strains without sequencing each genome, allowing comparison of genetic patterns from different strains. The first C. jejuni-specific microarray was generated from cloned DNA of NCTC11168 (Dorrell et al., 2001).

The basic mechanism of expression microarrays

Expression microarrays can examine numerous amounts of genes simultaneously. here we discuss the basic methodology of expression such as :

01. Extracting the RNA

The first step of the expression of microarray is extracting the RNA from desired cells or tissue and then running the lysate through the extraction column. in the extraction column, RNA molecules bind the column material. then add illusion buffer then RNA was illuted and finally pure RNA was got

This RNA was added to the reverse transcriptase PCR to be converted into cDNA. The cDNA was loaded into microarray chips (shown in Figure 1). In the microarray chip the cDNA loaded a differentially labeled these labels are florescent labels that use florescent dyes such as Cy3 or Cy5 to detect on the microarray

02.  Probe Design:

In this process uses a short fragment of DNA which is called a probe also named as cDNA clones or oligonucleotides. This probe is complementary to the selected gene. in expression, microarrays use probes which are typically short fragments of DNA which also called as oligonucleotides or cDNA clones, which are complementary to a specific gene of interest. glass slide or nylon membrane such solid surface used to observe the probes. several methods involve designing probes with computer algorithms, that predict the sensitivity and specificity of the probes. The cDNA hybridized to an array of probes.

03. Hybridization:

The cDNA hybridized to an array of probes. In the hybridization process above said labeled cDNA, in to the probe on the microarray as a complementary. Then slide washed to  remove unbounded cDNA.

04.  Scanning and Data Analysis:

The last step of the expression of the microarray process is to use a fluorescence scanner to scan the microarray to measure the signal intensity from each spot on the array. For the corresponding probe, the signal intensity is proportional to the amount of labeled cDNA hybridization. To compare gene expression levels between different samples or conditions use data analysis methods such as statistical analysis and visualization techniques which are used to know the differentially expressed genes or patterns of gene expression.

cDNA microarrays

• cDNA is a short form of complementary DNA which is synthesized from a RNA single stranded template, this process catalyzed by reverse transcriptase enzyme.
• cDNA is used to express a specific protein in a cell that does not normally express that protein or to sequence or measure mRNA molecules using DNA-based methods.

The cDNA microarrays are first developed by  Pat Brown at Stanford University which is a high-throughput technology simultaneously we can use to measure the expression patterns of thousands of genes. cDNA microarrays are a type of gene expression profiling technology that allows researchers to measure the expression patterns of thousands of genes simultaneously.

Figure 1 The principle of cDNA microarray (www.sciencedirect.com, n.d.)

Early expression arrays developed in 2000 were two-color arrays of limited resolution and contained approximately 5000 cDNAs. These arrays are mostly homemade and lack proper quality control measures, leading to problems in standardization.

Affymetrix single-color arrays

Affymetrix single-color arrays are a type of microarray technology that uses photolithography to create high-density microarrays on a single chip. In contrast to cDNA microarrays that use two different fluorescent dyes to label the test and reference samples, Affymetrix single-color arrays use one dye to label the test sample (typically Cy5).

The procedure for using Affymetrix single color arrays is as follows: First, the test RNA sample is reverse transcribed into cDNA and then transcribed into siRNA using T7 polymerase. The siRNA is then fragmented, labeled with a fluorescent dye (Cy5), and hybridized to the microarray. After hybridization, the microarray is washed to remove unbound cRNA and then scanned with a laser to determine the intensity of the Cy5 signal at each spot on the array.

Hybridization times for Affymetrix single-color arrays are generally shorter than for cDNA microarrays, typically taking about 8–16 hours to produce a reasonable amount of hybridization. The high-density nature of microarrays allows detection of gene expression levels for thousands of genes simultaneously.

A major advantage of Affymetrix single-color arrays is their ability to provide highly reproducible and accurate gene expression data due to the use of standardized protocols and quality control measures. However, a potential drawback of this technology is the high cost of microarrays and associated reactors, which may limit its accessibility to some researchers.

Advantages of Expression Microarrays

The high throughput capabilities of expression microarrays is one of their main benefits. Microarrays give researchers the ability to simultaneously analyze the expression of thousands of genes, giving them a thorough understanding of the patterns of gene expression in a single experiment. This is especially helpful for scientists researching diseases or complex biological systems because multiple genes may play a role in the underlying causes.

This method is one of the most cost-effective than next-generation sequencing and which is widely available and the cost per sample is much lower than others. This enables more broader use of this technology and makes microarrays accessible to researchers with constrained financial and material resources.

Since they have been in use for more than 20 years, microarrays have proven to be reliable and repeatable. Researchers can easily use microarrays in their experiments since standardized protocols and analytical techniques are publicly accessible. Researchers can also compare the outcomes of other studies or validate their conclusions using a multitude of publically accessible microarray data.

Disadvantages of Expression Microarrays

These days, microarray usage is declining due to the cost-effectiveness of RNA sequencing (Jaksik et al., 2015). Microarrays depend on prior genome knowledge for probe design, relying on known gene sequences. Probe performance hinges on gene annotation accuracy and completeness, impacting the reliability of results. Incomplete transcriptome coverage and missing significant genes can occur due to knowledge gaps or inaccuracies. RNA sequencing, on the other hand, does not require prior genomic knowledge, making it advantageous. It offers a more comprehensive view of gene expression and is suitable for poorly annotated genomes or non-model organisms. The shift towards RNA sequencing reflects its superiority in uncovering the complexities of gene regulation and expression.

Microarrays have several significant drawbacks. They are expensive per test and often rely on low-specification study designs. Lack of control over analyzed transcripts is common as most platforms use a set of manufacturer-designed probes. Microarrays exhibit relatively low accuracy, precision, and specificity. They are highly sensitive to variations in hybridization temperature, genetic material purity, degradation rate, and amplification process. These factors may affect the reliability of gene expression assays (Jaksik et al., 2015).

limited dynamic range

One of the major limitations of expression microarrays is their limited dynamic range. The sensitivity and signal intensity of the probes limit the spectrum of gene expression that can be accurately identified by microarrays. This means that at or near saturation levels, microarrays cannot detect low-abundance transcripts or highly expressed genes.

high background noise

When the expression of the microarrays process which produces high background noise this is one of the drawbacks of this kind of analysis. Background noise is a term used to describe the intensity of probes that is not due to specific binding. Bias in measuring relative fluctuations in the amount of DNA or RNA in biological samples results from failure to account for background noise. (Sui et al., 2009)

Cross-hybridization:

It is another significant limitation of expression microarrays. Cross-hybridization” in nucleic acids refers to base pairing between different sequences, forming double-stranded DNA, RNA, or DNA/RNA hybrids. This phenomenon is a disadvantage in research, potentially causing inaccurate gene expression analysis. Cross-hybridization can result in false positives and false negatives, undermining the reliability of genetic studies. Careful design is crucial. Researchers must account for cross-hybridization to ensure the accuracy and validity of their nucleic acid-based experiments.

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