7 Effects of dam nutrition during pregnancy on gene expression in the late-‐
7.3 Materials and methods 110
7.3.4 RNA methods 111
7.3.4.1 RNA extraction
Cryo-‐vials containing mammary biopsy tissue were removed from -‐80°C and stored on dry ice. Entire frozen biopsy samples were ground in 300 µl of Trizol reagent (Invitrogen, CA, USA) with a disposable, sterile pestle until partially homogenised. Trizol® reagent is a mono-‐phasic solution of phenol and guanidine isothiocyanate used to maintain the integrity of RNA while disrupting cells and dissolving cellular components. Once partially homogenised an additional 700 µl of Trizol® was added and further homogenisation was performed with a sterile 25 gauge needle and 1 ml syringe. The solution was left for 5 mins at room temperature (RT) to allow complete dissociation of nucleoprotein complexes, after which 200 µl of chloroform was added and mixed by vortex for 15 sec. The solution was left to incubate at RT for 5 mins. The samples were then centrifuged (Thermo Electron Corporation Heracus pico 17 centrifuge, Thermo Fisher Scientific, USA) at 12,000 g for 15 mins at 4°C, to separate the solution into aqueous (RNA-‐containing) and organic phases. A total of 400 µl of the aqueous layer was removed and added to an equal volume of 70%
ethanol and mixed with the pipette tip. A Qiagen RNeasy kit (Qiagen, Netherlands) was used to further purify the RNA. RNeasy kits are designed for fast purification of high-‐quality RNA using silica-‐membrane RNeasy spin columns which are able to bind up to 100 µg of RNA. A total volume of 700 µl of the RNA-‐containing aqueous solution and 70% ethanol mixture was added to an RNeasy column which was then centrifuged at 12,000 g for 15 sec to bind the RNA. The flow through was discarded. The column was then washed with 350 μl of RW1 buffer using centrifugation at 12,000 g for 15 sec. This step was repeated and the flow-‐ through was discarded. A volume of 500 µl of RPE buffer was added to the column and washed through using centrifugation at 12,000 g for 15 sec. Another 500 µl of RPE buffer was added to the RNeasy column and centrifuged for 2 mins at 12,000 g. The column was then placed in a sterile 1.5 ml eppendorf and 30 µl of diethylpyrocarbonate-‐ (DEPC) treated H20 was added to the centre of the column membrane and left to incubate for 1 min at RT.
The samples were then centrifuged for 1 min at 10,000 rpm to elute the RNA into a final collection tube. An aliquot was taken to check the quantity and quality of RNA extracted. The remaining RNA was stored at -‐80˚C.
7.3.4.2 RNA quantification
The concentration of RNA was determined using either the NanoDrop ND-‐1000 spectrophotometer (Thermo Scientific, MA, USA) or the Qubit 2.0 fluorometer, using the Qubit RNA assay kit (Invitrogen).
The Nanodrop measures the absorbance of an RNA or DNA sample at 260 nm and provides the concentration in ng/µl. The NanoDrop also assess the purity of each sample by measuring the A260 nm/A230 nm and A260 nm/A280 nm ratios. An A260:A280 nm ratio of
approximately 2.0 is generally accepted for pure RNA. Pure DNA has a ratio of around 1.8. Samples which have ratios below 2.0 may have high levels of proteins or residual phenol. The A260:A230 nm ratio should be between 2.0 and 2.2 for both RNA and DNA. Ratios below
this suggest the presence of contaminants such as EDTA, carbohydrates or phenol, which absorb at 230 nm. Trizol® reagent will absorb at both 230 and 270 nm therefore any remaining Trizol reagent will affect these ratios.
The Qubit uses fluorescent dyes to quantify biomolecules of interest. The fluorescent dyes only emit a signal when they are bound to specific target molecules (e.g., RNA). The Qubit has an advantage over the Nanodrop in that the fluorescent dyes are specific to the type of molecule being measured (Bustin et al., 2009). Therefore, even if there is DNA present in a sample, only the concentration of RNA will be given.
7.3.4.3 RNA agarose gels
The integrity of total RNA in samples was checked by running a small volume on a 1% agarose gel for quality control. Agarose gels were made with sodium boric acid buffer to which 50 μl of 10 mg/ml ethidium bromide stock solution was added giving a final concentration of 0.5 µg/ml. Approximately 1 µl of loading dye (25% bromophenol blue (1% solution), 25% xylene cyanol (1% solution), 30% glycerol and 20% MilliQ H20) was added to
each sample, then 10 µl of each sample was loaded on to the gel. A DNA ladder (1 kb+,
Invitrogen) was also loaded to one lane as a marker to determine the size of bands and as a control to check the quality of the gel. Gels were run for 10 to 20 mins at 2000 V (as RNA begins to degrade if run for longer periods). Gels were run with Owl B1A EasyCast Mini Gel systems (Thermo Scientific) using an EC250-‐90 power supply (Thermo Scientific). Gels were viewed under ultraviolet light and RNA checked for degradation. Intact total RNA should have sharp 28S and 18S rRNA bands; the 28S band should be approximately twice the intensity of the 18S band (indicating a 2:1 ratio). If RNA is partially degraded, the rRNA bands will be smeared in appearance, rather than sharp, or will not have the 2:1 ratio. Completely degraded RNA will appear as a low-‐molecular-‐weight smear.
7.3.4.4 RNA pools
The findings of Martin et al. (2012) and those reported in Chapter 5 demonstrated that dam nutrition during early pregnancy, as opposed to mid-‐to-‐late pregnancy, affected mammary gland development and first-‐lactation performance of offspring (Paten et al., 2013). Therefore, in the present study, only samples from ewes born to dams which were differentially fed during early pregnancy and fed maintenance during mid-‐to-‐late pregnancy (SmM, MM and AdM) were used for transcriptome analysis. For RNA-‐sequencing, RNA from multiple individuals within a treatment and time point was pooled in an attempt to minimise variation among samples (Kendziorski et al., 2003; Kendziorski et al., 2005; Konczal et al., 2014). Approximately 2 µg of RNA, subsampled from three randomly selected animals per treatment, was incorporated into pools. Three pools per treatment were generated for samples taken during late pregnancy and two pools were generated per treatment for samples taken during lactation, due to the reduced number of lactation samples. The pools were: late pregnancy; SmM, MM, and AdM (n = 3 pools sequenced for each treatment, with three samples per pool, n = 9 total samples for each treatment), and lactation; SmM, MM, and AdM (n = 2 pools sequenced for each treatment, with three samples per pool, n = 6 total samples per treatment). Therefore, the number of pools used to examine gene expression in late pregnancy versus lactation (irrespective of treatment) was as follows: n = 9 pools for
late pregnancy, with three samples per pool, n = 27 samples; and n = 6 pools for lactation, with three samples per pool, n = 18 samples. A diagram of the pooling strategy can be found in Chapter 8 (Figure 8.1).
7.3.4.5 Bioanalyzer
The quality of pooled RNA samples was checked using the Agilent 2100 Bioanalyzer with the Agilent RNA 6000 pico chip (Agilent Technologies, CA, USA). The bioanalyzer is a microfluidics-‐based system which utilises capillary electrophoresis to separate RNA molecules based on size. The RNA chip contains wells in which the samples are loaded. Micro-‐channels fabricated in glass create interconnected networks among the wells in the chip. When the chip is prepared, the micro-‐channels are filled with a fluorescence-‐gel-‐dye mix. Once the wells and channels are filled, the chip becomes an integrated electrical circuit. When the chip is placed in the bioanalyzer, a 16-‐pin electrode fits into the wells of the chip and charged biomolecules (e.g., RNA) are electrophoretically driven by a voltage gradient. Because of a constant mass-‐to-‐charge ratio and the sieve-‐like activity of the gel, the molecules are separated by size, with smaller fragments migrating faster than larger ones. Dye molecules complex with RNA strands and are detected by laser-‐induced fluorescence. Data is then translated into gel-‐like images (with bands) and electropherograms (peaks), which are interpreted much like a conventional gel electrophoresis (i.e., Intact samples have a 28S and 18S rRNA bands with a 2:1 ratio). The RNA integrity number (RIN) is derived through the bioanalyzer software and determines the amount of degradation present in each sample (Schroeder et al., 2006). A RIN of seven or greater (RIN ranges from 1, totally degraded, to 10, intact) is deemed acceptable for sequencing as it indicates little degradation.