title: SSH-PCR SON-C-D array
status: Public on Dec 02 2005
contact.city: Bristol
contact.first: DAVID
contact.last: MURPHY
contact.state: AVON
contact.address: Whitson Street
contact.department: Medicine and Dentistry
contact.country: United Kingdom
contact.institute: University of Bristol
organism: Rattus norvegicus
manufacture_protocol: Generation of SON subtracted cDNA libraries - We generated two subtracted libraries enriched for sequences in either control (C) or dehydrated (D) SON total RNA using the SSH-PCR technique (PCR-Select cDNA Subtraction Kit, BD Biosciences Clontech, Oxford, UK) Total RNA was extracted from pools of 10 -12 SON from either C or D animals using TRIzol (Invitrogen, Paisley, UK). Full-length double stranded cDNA, synthesised using the SMART kit (BD Bioscences Clontech, Oxford, UK), was digested with Rsa I to generate short, blunt-ended fragments optimal for adapter ligation and subtraction. The cDNA in which the specific transcripts are to be found is termed the “tester”, with the reference cDNA being the “driver”. Aliquots of both the C and D cDNAs were then ligated to either of two different adapters (1 or 2R) to generate tester populations (C1, C2R, D1 and D2R). To enrich for differentially expressed sequences present in either C or D SON mRNA populations, two sequential hybridisation reactions were performed. In the first hybridisation, an excess of driver cDNA was added to each tester. Following denaturation, the samples were allowed to anneal for 8 hours at 68 C. As annealing is faster for more abundant mRNA species, due to second order hybridisation kinetics, remaining single stranded tester molecules are enriched for low abundance sand differentially expressed sequences. The two samples from the first hybridisation were then mixed in the presence of fresh denatured driver cDNA and incubated overnight at 68C. This further enriches for differentially expressed sequences through the generation of double-stranded tester molecules with different single-stranded adaptor sequences on each end. Following dilution, hybridisation mixes were subjected to PCR (Advantage PCR Kit, BD Bioscences Clontech, Oxford, UK) which firstly fills in the adapter ends, creating binding sites for primer annealing. Only molecules with two different primer annealing sites are exponentially amplified. A subsequent nested PCR step further reduced background and enriched for differentially expressed sequences.PCR products were then cloned into pT-Adv Vector (Advantage PCR Cloning Kit, BD Bioscences Clontech, Oxford, UK) to produce two subtracted libraries enriched in differentially expressed cDNAs. Optimal ligation efficiency was obtained using <1 day old PCR product with a 1:1 vector:insert ratio. The reaction was incubated overnight at 14 C. The pT-Adv vector contains a single 3' T-overhang that is promiscuous in ligating all PCR products.  It also contains flanking M13 forward and reverse primer sites with the whole vector being resistant to kanamycin and ampicillin with a lacZ gene allowing simple blue / white visual identification for positive recombinant clones. Ligation mixes were transformed into TOP10F' E. coli competent cells using a modified heat shock method. White colonies representing true recombinants were picked from each plate, amplified and stored at -80 C.
  
  Microarray analysis of the cDNA libraries generated by SSH - The putative differentially expressed clones were arrayed on a nylon matrix (27) to generate the SON-C-D array. cDNA inserts were amplified in a 96-well plate format using PCR. The PCR products were checked by gel electrophoresis the transferred to 96-well 'V' bottom plates, ethanol precipitated and air dried overnight.  Pellets were resuspended in 40ml of 1xTE with 0.1M of NaOH added to immediately prior to arraying in order to denature the samples. Nytran+ supercharge (Schleicher and Schuel BioScience, Dassel, Germany) nylon membrane was coated with Krylon repositional adhesive (Schleicher and Schuel BioScience, Dassel, Germany) and cut to the correct size, trimming the edges with a razor and wearing gloves at all times.  The membrane units were placed into the arrayer, with enough to fill the entire plate.  This allowed 42 microscope slide size areas to be printed. Printing of the cDNA microarray was performed using an Affymetrix 417 (Affymetrix, Santa Clara, USA) arrayer.  This machine is capable of arraying 1152 spots in duplicate, producing 42 identical arrays in 8h of continuous printing. cDNA was spotted using a 300mm pin and spot spacing of 665mm centre to centre in 16 identical 12x12 grids.  After spotting, membranes were individually numbered before UV-cross-linking twice using a Stratalinker 2400 (BD Bioscences Clontech, Oxford, UK) at 120mJ/cm2.  As a quality control measure, arrays were inspected by sight, removed and baked at 70 C for 1-2h before being stored at -20 C in heat-sealed plastic bags.
  Separate microarrays were probed three times using independently generated target from either C or D SON. For each replicate, SONs from 12 rats, micro-dissected from 1-2 mm coronal slices, were pooled for RNA extraction. Radiolabelled target was generated from 3-10mg of total SON RNA (from either C or D rats) template. [±-33P] CTP was incorporated into cDNA using Superscript II reverse transcriptase (Invitrogen, Paisley, Scotland) and10-20-mer poly(dT) primer (Invitrogen, Paisley, Scotland). Arrays were placed DNA side inwards into 50ml disposable Falcon tubes and washed with 50ml of 2xSSC.  After draining of SSC and removal of air bubbles, membranes were pre-hybridised for 4h at 50 C with 4.0ml Microhyb (Invitrogen, Paisley, Scotland) containing 10ml of 1mg/ml human Cot 1 DNA and 10ml of 8mg/ml poly(dA) both denatured at 95 C for 5min prior to use.  The labelled target was denatured for 5min at 95 C, then added to the 4.0ml pre-hybridisation solution and allowed to hybridise to the probe at 50 C for 12-18h. Washing of the membranes was performed in 50ml 2xSSC 1.0% (v/v) SDS at 50 C, followed by 2x15min washes in 2xSSC 0.1% (v/v) SDS at 50oC.  Moist membranes were aligned on a plastic plate, wrapped tightly in plastic and exposed to phosphorimager screens for 1-3 days.  Spot intensities were captured into ImageQuant software (Amersham Biosciences, Little Chalfont, UK) by scanning screens at 50mm resolution on a phosphorimager (STORM; Amersham Biosciences, Little Chalfont, UK).