4. Methods and Materials
4.5.2. RNA quantification using a UV-visible spectrophotometer
RNA quantification was performed on the Thermo Scientific Nanodrop 2000 spectrophotometer instrument. Spectrophotometry determines the ability of a chemical substance to absorb light; every chemical compound and molecule absorbs, reflects or transmits light particles when measured over a certain wavelength - spectrophotometry
128 | P a g e records the samples absorbance. UV-visible spectrophotometers measure absorbance of the RNA samples over the ultraviolet range of 184-400 nm and the visible range of 400-700nm of electromagnetic radiation spectrum. This absorbance is then converted into a concentration value [211].
After calibration and measurement with a blank, 1μl of sample was placed onto the pedestal and measured at 260nm (A260), purity values were checked and concentration values recorded. Concentration values were obtained in triplicate for each sample, with only a variation of 0.5ng/μl allowed between values.
4.5.2.1. Advantages to UV-VIS spectrophotometry
The advantage of UV-VIS spectrophotometry is that there is minimal sample wastage, concentration values are obtained quickly with minimal training required, it is cost effective due to lack of additional reagent requirement and analysis includes evaluation of the relative purity of samples [212]. Another benefit is that there is no direct manipulation of the samples being analysed therefore the integrity is not compromised.
4.5.2.2. Disadvantages UV VIS spectrophotometry
The disadvantages with this technique is that accuracy can be effected by a number of contaminants; if total RNA extraction eluted DNA, proteins, chaotrophic salts or other complexes such as carbohydrates it will cause an incorrect value will be determine. UV VIS spectrophotometry cannot distinguish between the target of interest and contaminants that absorb light at an equal wavelength, since all nucleic acids display peaks at approximately 260nm all DNA or RNA molecules will contribute to the accumulated total absorbance at 260nm. Accuracy can also be effected by impurities within the samples reflecting light, since all light reaching the detectors is recorded this reflection off impurities would cause inaccuracy in concentration calculation.
Accurate light absorption is also controlled by the environment within the samples, absorbance is heavily influences by pH and temperature, which if combined with impurities will alter the absorption properties resulted in an incorrect concentration value for that sample. UV VIS spectrophotometry also displays inaccurate results at low concentration
129 | P a g e samples, due to the high concentration with vaginal material this was not deemed a highly contributing factor to quantification [212].
Determination of Semen through Haematoxylin &
Eosin Staining
When analysing semen samples the presence of spermatozoa must be confirmed, this is to ensure that any expression seen can be identified as coming from a ‘standard’ sample. A normal sample can be defined as having more than 20 million spermatozoa per millilitre of semen [213], this cannot be confirmed using a standard microscope however the identification of a large presence of semen is sufficient for this study. In the case of vasectomised samples the absence of spermatozoa must be confirmed; if spermatozoa are found then the miRNA expression will not distinguishable from the other semen samples collected.
The presence or absence of spermatozoa can be confirmed using the Haematoxylin and Eosin (H&E). It is a widely used staining technique and utilises two dyes. Haematoxylin is referred to as a basic dye (when mixed with a mordant such as an aluminium salt that help it bind) that stains basophilic or acidic structures, such as the nucleus a purplish-blue colour.
The mordant binds to any basophilic structure which is followed by the binding of the haematoxylin to the mordant. Eosin is a negatively charged, acidic dye which stains acidophilic or basic structures, such as most of the proteins in the cytoplasm, with a reddish pink colour. The combination of these two dyes provides a stain that highlights the nucleus and the areas of the cytoplasm where RNA is present in purple with the remainder of the cytoplasm being pink [214, 215]. In the case of staining semen, the basophilic heads of the spermatozoa will stain a purplish blue and thus make them detectable under a high powered microscope as seen in Figure 4.3.
Semen samples (1μl) were placed on labelled glass slides. Each slide was then placed in haematoxylin dye for 15 seconds before being gently rinsed around the stain with tap water. The slide was then placed into the eosin stain for two minutes followed by a gentle
130 | P a g e rinse around the stain. A cover slip was then placed over the stain and viewed under a microscope using 100x total magnification.
Figure 4.3 A micrograph of a typical semen stain using H&E (1) [215] and a micrograph displaying the complete absence of spermatozoa in the vasectomised sample provided by volunteer 63 following H&E staining (2).
Stem-loop Reverse Transcription alongside qPCR for miRNA Analysis
When analysing individual miRNA an extremely sensitive and reproducible method is needed. MicroRNAs range from 18 to 22nt in length with standard and quantitative PCR methods requiring a template that is a minimum of two times the length of either of the specific forward or reverse primers, each typically 20nt in length. Thus, the target minimum length is ≥40nt, meaning miRNAs are too short for standard RT-qPCR methods [216]. The solution to this problem is stem-loop reverse transcription followed by qPCR (RT-qPCR).
RT-qPCR involves several components that are optimised for miRNA specificity and sensitivity. The RT-primer contains a highly stable stem-loop structure that lengthens the target miRNA to form complementary DNA (cDNA). The forward PCR primer adds additional length with nucleotides that optimize its melting temperature and enhance assay specificity. The reverse primer disrupts the probe to produce fluorescence. As previous described, specificity can be improved by placement of the probe over much of the original miRNA sequence. The disadvantage to this strategy is that multiple high cost probes would be required to analyse multiple miRNA targets. The solution is to incorporate a universal
2
131 | P a g e probe sequence into the synthesised stem- loop RT primer which allows for a probe to be designed that can analyse any target marker. The T(m) of the probe is optimised by addition of a minor groove binding (MGB) complex [216].