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Microarray

A microarray is a technique to detect changes in gene expression of thousands of genes in parallel.

Microarrays are usually used to analyze the transcriptome of a sample. The transcriptome of an organism is the entirety of all genes expressed during its life time including embryogenesis. A microarray relies on specific probes, which are short pieces of chemically synthetized DNA complementary to the gene of interest. For each gene present on the microarray several little spots with the probe are attached to a slide. That means if the transcriptome of an organism is about 25.000 mRNA, the array would have about 100.000 spots in total, if four spots are printed per gene.

To analyze the transcriptome of a sample, the RNA needs to be isolated, reverse transcribed into cDNA (see also qPCR) and labeled fluorescently. Because the probe can bind extremely specifically to its target mRNA, the fluorescent labeled cDNA can now be hybridized to the microarray. After binding, the microarray can be washed and imaged using an automated fluorescent microscope. Because each spot contains some billions of probe molecules, the fluorescent signal intensity does not only tell whether a gene is expressed but also how much. Because each potentially expressed gene is present as probe on the microarray, the activity of virtually all genes in the sample compared to the control can be accessed at once.

The signals are then normalized to controls such as house keeping genes, which allows quantification and comparison of different samples. Microarrays have been proven to be an extremely versatility and useful tool in molecular biology.

DNA-Sequencing

DNA-Sequencing is a method to determine the sequence of a DNA molecule, which was developed by Frederick Sanger in 1977.

It requires a small piece of know sequence in the DNA molecule of interest to which the primer binds and from which the sequencing starts. If the entire DNA molecules is unknown, it can be pasted into another known DNA molecule that can be propagated in bacteria (i.e. DNA cloning). The steps of a sequencing reaction is similar to a PCR-reaction (denaturation, annealing, extension), but with just one primer, which results in a linear instead of an exponential amplification. In addition to the standard four nucleotides A, T, G and C, four modified nucleotides (dideoxy-nucleotides) are added in low concentrations, which will prohibit the further the extension of the newly synthesized DNA molecule, when incorporated. Because the incorporation of the modified nucleotides is at random, many DNA molecules of all sizes ranging from few base pairs to hundreds of base pairs in length will be generated. Consequently, the modified nucleotide is always at the end of each of the DNA molecules. Each of the modified nucleotides is labeled with a unique color (fluorophore), which can be detected by its fluorescence. Because all fragments of a specific length have the same labeled nucleotide at their end, the base at this specific position can be determined by its color. To determine the sequence of the DNA, the sequencing reaction is electrophoretically separated. The small fragments will reach the place where the fluorescence is measured first, the largest fragments last. Each fragment of a specific length will yield in a fluorescence peak, which tells what base is at the respective position of the sequenced DNA molecule.

Next Generation Sequencing – NGS

NGS is a method that allows the sequencing of millions of DNA templates in parallel in a cost-efficient and fast manner.

Because many different NGS protocols from different companies are around and due to the complexity of the sample preparation and sequencing process, only one widely used NGS system (Illumina®) is explained here.

The DNA of interest is first chopped in to smaller pieces of a relatively uniformed size and are ligated with adaptors to which the primers will bind. These fragments (the library) are attached to a solid carrier for example a glass slide. The billons of different DNA fragments are then clonally amplified on the carrier. Sequencing is performed in real-time by adding a DNA polymerase and reversibly fluorescently labeled nucleotides. For each base of the millions of different templates that is to be sequenced, the polymerase will add the respective nucleotide. Then the whole glass slide is excited by laser light, which will lead to a fluorescent signal for each newly added base of each fragment (as for Sanger sequencing, the four bases again have four different colors) and a picture is taken. Then the label is removed and the process is repeated many times. The resulting reads are between 50 and 250 base pairs. The total amount of sequence is around some billions of base pairs, which are highly redundant. The reads are assembled usually using a reference sequence to which they are mapped.

RNA-Seq

RNA-Seq ist used to analyze the whole transcriptome of a sample (e. g. cells, tissue etc.) by next generation sequencing (NGS).

The transcriptome is the sum of all expressed RNAs of a sample, which can be coding (mRNAs) and non-coding. To analyze the transcriptome of a given biological sample. the RNA is isolated and transcribed into cDNA. The cDNA library is then analyzed by NGS and the resulting sequences are assembled and mapped to a reference genome.