What are the methods used to measure and detect transcription?
Posted February 23, 2023
The most widely used methods for measuring and detecting transcription are Northern blot, qPCR, DNA microarray, RNA-Seq, and In-situ hybridization. An overview of each of the methods and the pros and cons of each:
Northern blot is a standard technique used for determining the quantity and size of a specific RNA in a sample. The advantages of Northern blot are that it’s a simple, low-cost procedure. However, it is time-consuming, requires a large quantity of starting material and precise oligonucleotide hybridization, and can only analyze a small number of samples at a time.
Quantitative Polymerase Chain Reaction (qPCR) uses a spectrophotometer to quantify transcription in real time. This technique offers several advantages including faster quantification of mRNA transcripts, ease of use, and the ability to use a multiplex approach to detect multiple mRNA transcripts. Limits of qPCR include the ability to quantify only a small number of transcripts in each reaction.
DNA microarray is capable of quantifying a large number of transcripts in a single experiment using nucleic acid hybridization of cDNA. The ability to quantify large numbers of transcripts in a single assay and its relatively low cost are the major advantages of this method. Its limitations include the inability to test multiple tissue samples in one experiment and the need for specialized equipment and software for image processing. In addition, because a control and a test tissue sample need to be prepared separately, microarrays are more time-consuming and could have a higher variance in the output data.
RNA-Seq works by sequencing RNA directly and counting the number of sequences. The major advantage of this technique is its ability to detect underlying genomic alterations at single-nucleotide resolution. Its high cost and the need for higher computational power and data-storage for downstream analyses are the two limitations of this method.
In Situ Hybridization
In situ hybridization (ISH) is a highly sensitive technique that is capable of detecting gene expression at a cellular level. It is widely used to detect nucleotide sequences in cells, tissue sections, and whole tissue. A major advantage of this technique is that it allows many different hybridizations to be performed on the same tissue, thus enabling maximum use of tissue that cannot be easily obtained. Difficulty in identifying targets with low RNA and DNA copies is a limitation of this method.
Probing steps in DNA transcription using single-molecule methods
Helixyte™ Green *20X Aqueous PCR Solution*
StrandBrite™ Green RNA Quantifying Reagent *200X DMSO Solution*