Project description:The 13,824 cDNA microarray was constructed by printing 6912 PCR-amplified cDNA clones in duplicate. These were derived from a previous EST study (Clark et al, 2007) and comprised 3840 clones from the Library D2 (fully dehydrated animals, cooled to -14°C) and 3072 clones from the Library D1 (dehydrated animals cooled to -2°C). The Stratagene SpotReport Alien Array Validation System (Stratagene, La Jolla, CA, USA) was included on the microarray. Construction and hybridization of the arrays were performed as described in Purac et al (2008) with the exception of the amino-modified primers used for the initial cDNA amplification for array printing, which in this study were: (pAL32FOR: TTCTCGGGAAGCGCG and M13 forward: GTAAAACGACGGCCAG). Hybridizations were performed using control animals in combination with the groups listed below.<br><br><br><br>Six groups of animals were used for the hybridizations. Treatments were as follows:<br><br>C = control = live animals from +5°C<br><br>M2 = cold dehydrated animals, cooled to -2°C at a rate of 2°C/week<br><br>M7 = cold dehydrated animals, cooled to -7°C at a rate of 2°C/week<br><br>H18 = animals taken from -7°C, and left to recover for 18 hours at 4°C with moisture.<br><br>Salt 0.9 = animals slowly dehydrated over a saturated solution of sodium nitrate (which gives a constant humidity of 98% RH) to produce animals with a similar water content as the M2 animals<br><br>Salt 0.2 = as for Salt 0.9, but the animals were dehydrated to a similar water content to the M7 animals.<br><br>6 biological replicates were performed for each condition, as well as some dye swaps.
Project description:The ability of the Antarctic microarthropod, Cryptopygus antarcticus (Collembola, Isotomidae), to survive low temperatures has been well studied at the physiological level. These investigations have indicated the importance of the moulting process in conferring this ability. This study investigated gene expression in groups of C. antarcticus that have distinct differences in their ability to survive low temperatures. A microarray containing 5,400 C. antarcticus expressed sequence tags was used to investigate gene expression differences between groups of animals with different supercooling points (SCP), and to low temperatures close to their SCP. By demonstrating the involvement of moulting genes in the differential survival of two groups of C. antarcticus with distinct SCPs, the results of this investigation add support to the role moulting plays in conferring cold tolerance in C. antarcticus.
Project description:High group animals with supercooling point (SCP) above -15°C and selected low group animals with SCP below -15°C were prepared from Cryptopygus antarcticus collected from wet moss (Sanionia uncinata (Hedw.)) during the austral summer of 2005 at the British Antarctic Survey's research station at Rothera Point, Adelaide Island (67'34'S, 66'8'W).
Project description:C3H/HeH females were mated with 101/H males<br><br>Mating was assessed by the presence of vaginal plugs the next morning. Identification of plug was designated day 0.5.<br><br> <br><br>At 8.5 days of development the dam was culled by cervical dislocation, the uterine horns disscted and placed into PBS. The uterus was dissected to release decuduae and these opened according to standard protocols. Morphologically normal looking embryos of between 4 and 6 somites were collected. The left and right lateral plates were dissected away with watchmakers forceps and pools of 4 left and 4 right lateral plates were snap frozen in LN2.
Project description:Direct comparison of gene expression in 13.5dpc embryonic gonad/mesonephros between male Mrotm1H/Mrotm1H mice and male wild-type littermates. Two independent pools of 10 gonads (5 mice) were compared on 4 microarrays (two colourswaps).
Project description:To generate differential gene expression profiles across the moult cycle of Antarctic krill, relative to an early intermoult stage.
Project description:Two groups of Laternula elliptica, old and young, were subjected to both ambient control and hypoxic conditions, and their gills were extracted and hybridized on an array.
Project description:Specimens of L. elliptica were collected by scuba divers at a depth of 10-18m in January 2010 at Hangar Cove, Rothera Point, Adelaide Island, Antarctic Peninsula (67°34’07°S, 68°07’30°W). Animals were collected in two size classes: large animals (with lengths ranging around 60mm) and small animals (lengths ranging around 30mm), the sizes of which equated to average ages of 16 and 7 years respectively. These two groups were termed “old” and “young” respectively. The clams were maintained in a flow-through aquarium and allowed to acclimate to laboratory conditions for 2 weeks before experimentation. At the end of the acclimation period, 10 old and 10 young animals were transferred to a 60l jacketed tank with aerated sea water, connected to a thermocirculator. The sea water temperature was gradually raised from 0°C to +3°C over a 12 hour period. This temperature was then maintained for a further 12 hours, before sampling the animals. Gill tissue samples were dissected and immediately snap frozen in liquid nitrogen and stored at -80°C. The sampling regime was repeated on 10 old and 10 young animals that had been maintained in the flow-through aquarium for the same time period (control animals) RNA was extracted from all samples using TriSure, according to manufacturer’s instructions. RNAs from gill from 5 animals from each group (old treated, young treated, old control and young control) were used in the array hybridization experiments. The array used was A-MEXP-1676. PCR amplified labelled cDNA targets were prepared from 1μg total RNA. 5 replicates were used with 3 dye swaps.