title: Tortula ruralis cDNAs from Suppressive Subtractive Hybridization
status: Public on Jan 10 2009
contact.city: Lubbock
contact.first: Paxton
contact.last: Payton
contact.email: ppayton@lbk.ars.usda.gov
contact.state: TX
contact.address: 3810 4th Street
contact.country: USA
contact.web_link: http://www.csrl.ars.usda.gov
contact.institute: USDA-ARS
contact.phone: 806-723-5218
organism: Syntrichia ruralis
description: We established a cDNA microarray containing the inserts (PCR derived fragment) from each of the 768 individual SSH ESTs, with the exception of thirteen that failed to generate a PCR fragment. Twelve of the missing thirteen SSH EST cDNAs were replaced with PCR fragments from previously isolated T. ruralis cDNAs four of which, representing the ribosomal proteins S14, S16, L23 and L15 (Wood et al., 2000, Zeng and Wood 2000), were previously reported to be constitutively expressed and were added to serve as normalization genes. The remaining eight clones, Tr155, Tr217, Tr403, Tr416, Tr421 (described by Scott and Oliver, 1994), and TrCDPK (U82087) were added as either positive “up-regulated” (Tr155, Tr403, Tr421), negative “down-regulated (Tr217, Tr416), or neutral (TrCDPK) controls based on previous northern analyses. The cDNAs were printed from two 384 well plates in 12 blocks (two columns of 6) of 24 x 8 spots such that each SSH EST and controls were represented in triplicate. Each of the triplicate cDNAs was separated within the blocks to eliminate possible spatial hybridization bias. All hybridizations were duplicated as dye swaps with two separate RNA preparations, from large populations of individual gametophytes (isolated from a minimum of three separate clumps), serving as the source for the sscDNA Cy3 and Cy5 labeled targets.  The RNA preparations for the Total polyA RNA were by necessity separate samples from those used to isolate Polysomal poly A RNA.
manufacture_protocol: T. ruralis ([Hedw.] Gaertn, Meyer and Scherb) gametophytes were collected and stored dry at room temperature. For experiments, gametophyte tissue was hydrated for 48h as described previously (Oliver, 1991; Scott and Oliver, 1994) to ensure recovery of the tissue to full activity from its exposure to drying environments in the field. These gametophytes were sampled and flash frozen to represent hydrated controls. Rapid-dried moss was prepared by placing fresh gametophyte on filter paper over activated silica gel in a petri dish as described previously (Bewley and Thorpe, 1974).  The rehydrated state was obtained by immediately flash freezing in liquid nitrogen following 2h of rehydration.  Slow-dried moss was prepared by placing fresh gametophytes of known weights on a nylon mesh in a closed chamber over a stirred saturated solution of sodium nitrite at 20˚C (RH 66%) for 24 h. Using this regime the air-dried weight (ca. 20% of original fresh weight) was obtained within 6h (Oliver, 1991).
  Two subtractive suppression hybridization libraries were constructed using a PCR-Select cDNA Subtraction Kit  (Clontech, Palo Alto, California, USA) following manufacturer’s instructions: Slow-dried Polysomal (SDP) and Rapid-dried Rehydrated polysomal (RDPR).  For each cDNA subtraction, hydrated control gametophytic poly (A+) RNA was used as the source for driver cDNA.  2μg of mRNA was the starting material for both tester and driver. The subtracted cDNAs were cloned into the T/A vector pUCR19r (Oliver et al., 2001) and transformed into DH5α cells (Invitrogen, Carlsbad, CA). Transformants were selected on LAXI plates (100μg/ml AMP, 0.008% X-Gal, 200μM IPTG) for blue/white screening.  White colonies were picked into 96-well plates containing 200μl of LB/AMP (100μg/ml) and allowed to grow overnight shaking at 37˚C.  80μl of LB:glycerol (1:1) was added to the wells (final concentration of 17% glycerol), and stored at –80˚C.
  PCR generated DNA fragments in 2.5X SSC, 0.1% sarcosyl at a concentration of 250ng/ul were arrayed in 384-well plates for printing.  Poly-L-lysine coated slides were printed using a GMS 417 microarrayer (Affymetrix, Santa Clara, CA, USA).  Three replicates for each fragment (2 hits/spot) were printed for each block, with two blocks per slide.  The slides were processed and the cDNAs cross-linked as described by Hegde et al., 2000 and stored in the dark at room temperature.