Investigation Title Expression Profiling to Identify Novel Desiccation Response Transcripts from Tortula ruralis gametophytes Comment[Submitted Name] Expression Profiling to Identify Novel Desiccation Response Transcripts from Tortula ruralis gametophytes Experimental Design transcription profiling by array Experimental Design Term Source REF EFO Comment[SecondaryAccession] GSE13680 Comment[ArrayExpressReleaseDate] 2010-05-06 Comment[AEMIAMESCORE] 3 Comment[ArrayExpressAccession] E-GEOD-13680 Comment[MAGETAB TimeStamp_Version] 2010-07-30 12:23:40 Last Changed Rev: 13058 Experimental Factor Name Experimental Factor Type Experimental Factor Term Source REF Person Last Name Dowd Oliver Hudgeons Payton Person First Name Scot Melvin Jeremy Paxton Person Mid Initials J Person Email sdowd@pathogenresearch.org ppayton@lbk.ars.usda.gov Person Phone 806-789-6879 806-723-5218 Person Fax 806-771-1168 806-723-5272 Person Address MBRI, MBRI, 4321 Marsha Sharp Fwy, Lubbock, TX, USA USDA-ARS, 3810 4th Street, Lubbock, TX, USA Person Affiliation MBRI USDA-ARS Person Roles submitter submitter Person Roles Term Source REF The MGED Ontology The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2010-05-06 PubMed ID Publication DOI Publication Author List Publication Title Publication Status Publication Status Term Source REF Experiment Description In this study we have tried to utilize the unique aspects of the T. ruralis response to desiccation and rehydration to design a strategy to identify rehydrins that are of low abundance and perhaps completely novel to the desiccated or rehydration transcriptomes. We have constructed two Subtractive Suppression Hybridization (SSH) libraries (Diatchenko et al., 1996) that are designed to enrich for differentially expressed low-abundance transcripts contained within gametophytic cells either in the slow-dried state (mRNP sequestrated rehydrin transcripts) or cells that have been rapidly dried, rehydrated and sampled at 2h of hydration (rehydrin and recovery transcripts) when the translational change in gene expression is at its peak (Oliver 1991). To achieve this aim we constructed SSH libraries using PolyA RNA isolated from the polysomal (mRNP) fractions from the slow-dried and 2h rehydrated rapid dried gametophytes selected against PolyA RNA from hydrated control gametophytes as the source for driver cDNA. Collections of cDNA clones from each library were sequenced and used to generate a small T. ruralis SSH cDNA microarray for expression profiling of both total RNA extracts for transcript accumulation assessments and polysomal RNA extracts for transcript sequestration and recruitment assessments. To assess the expression characteristics of the transcripts represented by the SSH contigs 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 probes. The RNA preparations for the Total polyA RNA were by necessity separate samples from those used to isolate Polysomal poly A RNA. Protocol Name P-GSE13680-5 P-GSE13680-16 P-GSE13680-9 P-GSE13680-13 P-GSE13680-6 P-GSE13680-14 P-GSE13680-7 P-GSE13680-15 P-GSE13680-8 P-GSE13680-10 P-GSE13680-11 P-GSE13680-12 P-GSE13680-17 P-GSE13680-3 P-GSE13680-1 P-GSE13680-2 P-GSE13680-4 Protocol Type specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action grow nucleic_acid_extraction nucleic_acid_extraction labeling labeling hybridization image_aquisition feature_extraction feature_extraction bioassay_data_transformation bioassay_data_transformation bioassay_data_transformation bioassay_data_transformation Protocol Description 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). Samples were flash frozen in liquid nitrogen. 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 and flash frozen in liquid nitrogen. 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 and flash frozen in liquid nitrogen. 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. Samples were flash frozen in liquid nitrogen. 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. Total RNA was isolated using a series of phenol extractions as described by Lane and Tumaitis-Kennedy (1981). The final nucleic acid pellet obtained by this procedure was resuspended in 5ml of 1 X TE buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA), 0.73g of NaCl was added, and the sample was placed at 4ºC overnight to precipitate RNA. This NaCl precipitation was repeated again prior to resuspension in 1 X TE for analysis or water for Poly A RNA fractionation. Total RNA was isolated using a series of phenol extractions as described by Lane and Tumaitis-Kennedy (1981). The final nucleic acid pellet obtained by this procedure was resuspended in 5ml of 1 X TE buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA), 0.73g of NaCl was added, and the sample was placed at 4ºC overnight to precipitate RNA. This NaCl precipitation was repeated again prior to resuspension in 1 X TE for analysis or water for Poly A RNA fractionation. Cy3 and Cy5 labeled probes for hybridization were made from 300-450ng of poly (A)+ mRNA using the 3DNA Submicro EX Expression Array Detection Kit (Genisphere, Hatfield, PA, USA) as described by the manufacturer. Hybridizations were performed at 60ºC in hybridization chambers (2551, Corning, Corning, NY, USA) under water using the 2-step hybridization protocol described by the manufacture. Cy3 and Cy5 labeled probes for hybridization were made from 300-450ng of poly (A)+ mRNA using the 3DNA Submicro EX Expression Array Detection Kit (Genisphere, Hatfield, PA, USA) as described by the manufacturer. Hybridizations were performed at 60ºC in hybridization chambers (2551, Corning, Corning, NY, USA) under water using the 2-step hybridization protocol described by the manufacture. Target cDNAs were hybridized at 60C for 12 hours in a water bath. Slides were washed 15 minutes at 60C in the dark in 2X SSC with 0.1% SDS, 15 minutes at room temperature in 2XSSC with 0.05% SDS, and 10 minutes at room temperature in 2X SSC. Slides were rinsed briefly in 100% ethanol and dried by centrifugation. Slides were scanned using an Axon4000B array scanner. Image analysis and spot quantification were performed with Imagen 4.2 software. Image analysis was performed with Imagene 4.2 (Biodiscovery, Inc). Selection criteria for "good" spots was as follows: 1) signal strength-processed signal (minus background) < 3 x standard deviation of the mean local background 2) signal uniformity-pixel to pixel signal variation < 20% 3) replicate uniformity-mean processed signal (minus background) must have a coefficient of variance < 0.2 4) replicate mean signal strength-one channel must have processed signal > 1.5% of the quantifiable maximum signal. Data analysis was performed using GeneSpring 5.1 (Silicon Genetics, Inc.). Signal was normalized to the 50th percentile of the positive control values. Image analysis was performed with Imagene 4.2 (Biodiscovery, Inc). Selection criteria for "good" spots was as follows: 1) signal strength-processed signal (minus background) < 3 x standard deviation of the mean local background 2) signal uniformity-pixel to pixel signal variation < 20% 3) replicate uniformity-mean processed signal (minus background) must have a coefficient of variance < 0.2 4) replicate mean signal strength-one channel must have processed signal > 1.5% of the quantifiable maximum signal. Data analysis was performed using GeneSpring 5.1 (Silicon Genetics, Inc.). Signal was normalized to the 50th percentile of the positive control values. ID_REF =
VALUE = normalized ratio Slow-Dried (SD)/Hydrated (HYD) polysomal RNA samples
VALUE_STDEV = standard deviation for the normalized ratio pSD/pHYD
t-test_P-value = t-test p-value
TREATMENT_SIG_MEAN = mean signal for the Slow-Dried sample from polysomal RNA
CONTROL_SIG_MEAN = mean signal for the Hydrated sample from polysomal RNA ID_REF =
VALUE = normalized ratio Slow-Dried (SD)/Hydrated (HYD) total RNA samples
VALUE_STDEV = standard deviation for the normalized ratio tSD/tHYD
t-test_P-value = t-test p-value
TREATMENT_SIG_MEAN = mean signal for the Slow-Dried sample from total RNA
CONTROL_SIG_MEAN = mean signal for the Hydrated sample from total RNA ID_REF =
VALUE = normalized ratio Rapid-dried Rehydrated(RDR)/Hydrated (HYD) total RNA samples
VALUE_STDEV = standard deviation for the normalized ratio tSD/tHYD
t-test_P-value = t-test p-value
TREATMENT_SIG_MEAN = mean signal for the Rapid-Dried Rehydrated sample from total RNA
CONTROL_SIG_MEAN = mean signal for the Hydrated sample from total RNA ID_REF =
VALUE = normalized ratio Rapid-dried Rehydrated (RDR)/Hydrated (HYD) polysomal RNA samples
VALUE_STDEV = standard deviation for the normalized ratio pRDR/pHYD
t-test_P-value = t-test p-value
TREATMENT_SIG_MEAN = mean signal for the Rapid-dried Rehydrated sample from polysomal RNA
CONTROL_SIG_MEAN = mean signal for the Hydrated sample from polysomal RNA Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology SDRF File E-GEOD-13680.sdrf.txt Term Source Name The MGED Ontology ArrayExpress EFO The MGED Ontology Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version