Componentes de un digestor anaeróbico

RNA concentration and purity were assessed using a spectrophotometer (NanoDrop™ _2000, Thermo Fisher Scientific, Denmark). One microliter of extracted RNA was pipetted onto the NanoDrop probe and the amount of ultraviolet (UV) light absorbed at 260 nm, the wavelength at which nucleic acids best absorb light, was measured by a photo-detector to determine the

concentration inferred using the Beer-Lambert law (see Equation 2.2). Following assessment of RNA concentration, the purity or RNA ‘quality’ was determined from the ratio of absorbance at 260 nm to 280 nm, the wavelengths at which RNA/DNA and protein best absorbs UV light, respectively. Highly purified RNA is indicated by a A260/A280 ratio of 2. Other potential contaminants include ethanol, phenol or guanidine, which are measured at 230 nm. Therefore, the A260/A230 ratio is also measured where a reading >1.5 of is preferred. RNA concentrations and purities are reported within the methods section of each experimental chapter throughout this thesis.

A= ЄlC Where:

A = absorption at 260 nm

Є = the molar extinction coefficient (40 µg/ml for RNA and 50 µg/ml for dsDNA) l = the path length of the spectrophotometer

C = concentration of RNA/DNA

Equation 2.2 Quantifying RNA and DNA concentrations using spectrophotometry

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

2.9.4! Principle

In order to produce functional proteins and therefore alter the muscle phenotype, a gene must first be transcribed in the nucleus to produce a messenger ribonucleic acid (mRNA) which is later translated into protein within the ribosome. The amount of a gene expressed typically (yet not exclusively) translates to the amount of protein produced which may influence the overall structure and function of SkM tissue. Assessment of mRNA regulation in response to a given stimulus has therefore become of particular interest in cellular and molecular

physiology/biology. Since DNA is a very large molecule with such limited copies, assessment of mRNA expression proved difficult until the 1980’s where polymerase chain reaction (PCR), a technique widely used to amplify specific fragments of the DNA, was introduced by Kary Mullis who later won the Nobel prize in chemistry in 1993 (Mullis & Faloona, 1987).

Following isolation of mRNA from tissue or cells (see sections 2.9.1 and 2.9.2), the single stranded RNA (ssRNA) must be reverse transcribed to form a complimentary DNA (cDNA) before the target sequence is amplified. In order for reverse transcription (RT) to occur, oligonucleotides (dNTPs) are added to the ssRNA using the enzyme reverse transcriptase and cDNA is synthesised from the 3’ to 5’ end of the mRNA molecule. Once synthesised, the DNA is amplified via three distinct steps, all of which represent 1 cycle of a PCR reaction that is repeated numerous times (30-40 cycles) to produce ~ 1 bn copies of the target sequence. These steps include: 1) Denaturation whereby the double stranded DNA (dsDNA) is subjected to high temperatures (95˚C) in order to separate the DNA into two strands exposing the 3’ end of the DNA. 2) Annealing of primers then occurs whereby the temperature is lowered (optimal temperature is primer specific, with all the primers used herein designed to anneal at approximately 60˚C) to enable binding of short sequence (approximately 18-30 bp) primers to the DNA strands. 3) Extension then takes place. Here, Taq polymerase, an enzyme derived from the bacterium species, Thermus Aquaticus that is able to withstand such high temperatures, binds to the primers and synthesises the complimentary strand using free dNTPs.

During each PCR cycle, a fluorescent dye or probe, binds to each dsDNA molecule after primers anneal to the 3’ end. The amount of light excited and emitted from the fluorescent molecule is able to provide a ‘real-time’ measurement of DNA amplification as the amount of light measured by a fluorometer within the PCR thermocycler instrument is directly proportional to the amount of targeted DNA produced. This can easily be quantified following

each PCR run, according to the amount of cycles required to exceed the fluorescence cycle threshold (CT) self-reported. Thus, generally, the lower the CT value, the higher the expression levels as the fluorescence being detected earlier above background fluorescence reflects the larger amount of starting nucleic acid material. Conversely, the higher the cycle number, the later detection of fluorescence above background levels reflecting a lower starting abundance of the nucleic acid material. The resultant CT values of the target gene in each sample are then compared to the CT values of a housekeeper/reference gene (one of which should remain consistent, regardless of any given stimulus) to determine either absolute or relative quantities (Schmittgen & Livak, 2008).

2.9.5! Procedure

Throughout this thesis, a one-step PCR kit (QuantiFast SYBR® _{Green RT-PCR Kit, Qiagen,} UK) was used to analyse mRNA expression whereby the cDNA synthesis and PCR steps were performed in the same reaction tube for time efficiency and reduced risk of cross contamination. Reaction tubes were either prepared manually by hand or automatically using the QIAgility robot instrument (Qiagen, Crawley, UK). Each reaction included 50% SYBR® Green master mix (containing HotStarTaq DNA polymerase, RT-PCR buffer, SYBR green fluorescent dye and ROX passive reference dye) 1% reverse transcriptase (RT) mix and 0.75% of forward and 0.75% reverse primer, with the remaining 47.5% as diluted RNA sample (7.37 ng/µl in nuclease-free H2O). The preparation method (i.e. manual or automatic using the QIAgility robot), alongside the specific volumes (µl) of PCR reagents and RNA sample used per reaction will be specified within the methods section of each experimental chapter. Prepared reaction tubes were then transferred to a PCR thermal cycler (Rotor-Gene 3000Q, Qiagen, UK) to undergo reverse transcription/cDNA synthesis (hold 50˚C for 10 min), transcriptase inactivation and initial denaturation (95˚C for 5 min) followed by 40 ×

amplification cycles consisting of; 95˚C for 10 s (denaturation), 60˚C for 30 s (annealing and extension).