Methods for expression analysis

Novel miRNAs are discovered via cloning, and northern blot is used to confirm their existence [187]. miRNAs can also be predicted by algorithms which search the genome for conserved sequences with structure similar to known miRNAs.

Since their discovery, various techniques have been developed which can allow us to analyse the localisation and quantity of miRNA transcripts being expressed in a particular cell line, tissue or organism. There are several challenges in determining miRNA expression: it is difficult to design specific probes or primers for miRNAS as their GC content varies widely leading to very different melting temperatures, and they are small and have high levels of homology within miRNA families. This is further complicated by the multiple stages of processing miRNAs go through; probes and primers must be specific to the mature miRNA and not one of the earlier stages, as pri- and pre- miRNAs are often expressed at drastically different levels than the corresponding mature miRNA. The mechanisms which control the amount of precursor molecules which are processed into active miRNAs are not well understood but they produce important functional effects [188]. The various methods which have been used to overcome these technical challenges are discussed below.

Microarrays are a useful way of quickly assessing hundreds of miRNAs: A slide is

printed with 1000s of oligos homologous to miRNAs and the RNA from the sample is conjugated to a fluorescent dye and allowed to hybridise to the oligos on the slide. The accuracy of microarray analysis is limited because of the short length and varying melting temperatures of the miRNA but this problem can be ameliorated in several ways. Altering the length of the probes can solve the problem of the differences in melting

temperature [189], but it can further reduce the specificity of probes which are already very short. Another solution is to chemically alter the probes to increase hybridisation stability, for example using locked-nucleic acids (LNAs) which allow hybridisation over a wider range of temperatures [190].

Probes are also printed multiple times in different places on the slide as this allows the

researchers to take an average which is more accurate, and reduces the problems caused if the slide gets scratched or contaminated with dust.

72

In spite of these efforts, microarrays are still limited in their accuracy and are often poor at distinguishing between mature miRNAs and their precursors. However they are still

frequently used as a first step in studying the role of miRNAs in disease, because they allow rapid screening of thousands of candidates. In these experiments a panel of thousands of miRNAs is used to compare diseased and healthy tissue and this comparison is used to suggest which miRNAs should be validated by other methods and further studied for their role in pathogenesis.

qPCR can accurately analyse the expression level of a miRNA: qPCR is often used to

validate specific miRNAs which have been identified as being of interest by microarray data. In qPCR the RNA in the lysed cells is converted to cDNA, PCR is then performed on this in the presence of a fluorescent DNA dye. The fluorescence level is measured during every PCR cycle which allows precise quantification of the target. A value is assigned to the sample based on the number of cycles it takes for the fluorescence to reach a particular level. This is known as the Ct value. qPCR can be used to assess the absolute or relative quantity of the target miRNA, in absolute quantification the Ct value is measured against a serial dilution of RNA of a known quantity and in relative quantification the Ct values of two samples are compared using a housekeeping small-RNA as an internal control using the ΔΔCT equation:

ΔΔCt = ΔCt,sample - ΔCt,reference

When performing qPCR on the mRNA for a gene, reverse transcription to create cDNA is performed using random short primers and/or oligo(dT) primers. However miRNA transcripts don’t have polyA tails and are much too short for the random oligo hexamers to bind to. The first widely used solution found was to create specific primers for reverse transcription of a particular target miRNA [10]. In order to work specifically in spite of the short transcript the primer is designed to form a stem loop, with the homologous region overhanging (see Figure

4.1). This solution is effective and allows specific amplification but it requires cDNA to be

made separately for each miRNA studied. This means that more sample must be used for each test, and reduces accuracy by increasing the chance of pipetting errors as well as being more time consuming for the researcher.

73

A more recent innovation [191, 192], allows a polyA tail to be enzymatically conjugated to the miRNA before reverse transcription. cDNA can then be made by reverse transcription can then be done using an oligo(dT) primer. qPCR remains the most accurate and specific method of quantifying the expression of an individual miRNA.

Other methods for miRNA analysis: In addition to these methods levels of miRNA

expression can be estimated using in situ hybridisation, which shows localisation in cells and tissues. The role of miRNAs in development has been studied using genetic modification to tie expression of a specific miRNA to a fluorescent marker, for example and cells which constitutively express Luciferase or GFP with 3’ complimentary sequence to miRNA of interest and a bioluminescent or fluorescent protein of another colour under the same promoter but without the miRNA binding region acting as an internal control [193, 194]. These methods are good for information on the localisation of miRNAs but are not

quantitative. Electrochemical biosensors for detection of miRNA are being developed as well [195], but recent advancements in RNA-Seq technology mean that it is now potentially viable to replace other methods such as microarray and qPCR [196].

Figure 4.1

Figure 4.1: Schematic showing how the two-step stem-loop Taqman miRNA RT-PCR system works. [10]

74

4.2 Microarray analysis comparing hematopoietic progenitors derived from hESCs or