Emma's+Lab+Notebook+Autumn+2010

__Ceramide__ Worked on analysis/manuscript/presentation.
 * December 16, 2010**

Updated lab notebook with links to qPCR data.

Ceramide sequences Successful sequences: Fas, Insulin receptor, Lass5, Leptin Trimmed sequences in Geneious to get rid of poor quality data. enter trimmed sequences into blastx, searching swissprot doing (1) a search with no a priori taxonomy and (2) a search qualifying crassostrea.

__Ceramide & vibrio exposure: qPCR results__ [|Link] to results Significant differences in expression between control and vibrio-exposed for both PE2 (p=0.00184) and Sptlc1 (p=0.009616). media type="custom" key="7858455" media type="custom" key="7858465"
 * December 15, 2010**

Also significant differences in expression of Sptlc1 between adult C. gigas tissues: gill-dig.gland: p=0.0032 muscle-dig.gland: p=0.0284 mantle-gill: p=0.0061 muscle-gill: p=0.00013 muscle-mantle: p=0.007 media type="custom" key="7858501"

__Vibrio exposure: qPCR of ceramide pathway and nutrition genes__ qPCR on pooled control and VE samples; in duplicate with remaining ceramide primers (Fas, Lass5, NGF), Hsp70, Insulin receptor, and leptin receptor. media type="custom" key="7858527" Results: [|Link] to results. Contamination in NTCs for Hsp70 so did not analyze those results. Ceramide pathway: Lass5 and NGF are expressed differently between treatments (p=0.015 and p=1.7e-4, respectively). Fas is not differentially expressed, but there are also large standard deviations on the technical replicates. media type="custom" key="7861679" media type="custom" key="7861689"media type="custom" key="7861693" Nutrition: Both insulin and leptin receptors are expressed at higher levels in the vibrio-exposed oysters (p=0.04 and p=7.4e-4). media type="custom" key="7861705"media type="custom" key="7861711"

__Ceramide: qPCR__ Tissue distribution of Sptlc1 (qPCR previously failed for this gene). Also for pooled control and vibrio exposed adult gill tissues (see below).
 * December 14, 2010**

__Vibrio exposure: qPCR__ Pooled control (n=10) and vibrio exposed (n=10) samples reverse transcribed Dec 8, 2010 that had previously been PCR'd separately. 10 uL of each sample into each pool. qPCR done for EF1a, prostaglandin receptor (PE2), and Sptlc1. media type="custom" key="7858371"

__Ceramide: Follow-up to gene verification__ (see Dec 6, 2010) Gene: Lass5 (LAG1 homolog ceramide synthase) SR returned glucose-regulated protein (Q16956). Alignment with C. gigas sequence for Lass5 (sequence used for primer design) had a lot of gaps - i.e. not a great alignment. Also a lot of gaps when aligned with original P. anubis sequence. Need to see what amplified product looks like before deciding to keep or discard.
 * December 13, 2010**

Gene: MHC SR returned myosin heavy chain...oops! wrong MHC? discard this one.

Gene: IL1B SR returned serine/threonin-protein phosphatase 2A (PP2A). Probably not IL1B, but is involved in apoptosis, so will keep for now.

Gene: Sptlc1 verified as serine palmitoyltransferase 1. Enzyme that catalyzes one of the first steps in ceramide synthesis.

Gene: Sphingomyelinase (SMase) Verified as acid sphingomyelinase-like phosphodiesterase 3b.

Gene: nerve growth factor B SR returned cation transport regulator-like protein, or Chac1. Chac1 is a pro-apoptotic component of the unfolded protein response pathway and may mediate some pro-apoptotic effects.

Gene: Fas death receptor SR returned TNF receptor superfamily, which plays similar role and is related to Fas receptors in ceramide-mediated apoptosis pathway.


 * December 10, 2010**

__NOAA OA: qPCR__ and __Vibrio exposure: qPCR__ qPCR of NOAA OA samples at Spltc1, Lass5, NGF, and Hsp70 (in duplicate). Sptlc1 for Vibrio exposure (duplicate). Used 3StepAmp+Melt_SJW protocol. media type="custom" key="7811395"

__ NOAA OA: qPCR Results __ [|Link] to results p-values are pairwise comparisons with 380 ppm treatment. ANOVA was non-significant. media type="custom" key="7814741" media type="custom" key="7814817" media type="custom" key="7814975"

__Vibrio exposure: results__ media type="custom" key="7815025" Notes for next time: If doing qPCR on samples of juveniles or adults that have RNA/cDNA for individuals, try to pool treatments and do qPCR on the pooled cDNA to decrease variability and pull out underlying trends/differences between treatments. Will also save reagents.

__NOAA OA: qPCR (SMase)__ with optimized protocol: 45 cycles, 58C annealing T media type="custom" key="7815381" Results ([|Link]): All samples amplified before cut-off of 45 cycles and had the same melt peak, but all NTCs had strong amplification of product so results are not useful.

__Ceramide: Sequencing__ Prepared DNA and primer plates for sequencing on Monday. Melissa Baird is filling the rest of the plate and will bring it down to the sequencing center. media type="custom" key="7815435"

__NOAA OA: qPCR__ and __Vibrio exposure: qPCR__ All 4 NOAA OA samples and n=5 controls and VE for vibrio (C1-5 and VE 1-5). Used EF1a and FAS; all in duplicate. media type="custom" key="7815367" Results ([|Link]): All negatives were clean and samples amplified. NOAA OA larvae FAS expression by treatment. There is 0 expression at 2000 ppm. Differences in expression are not statistically significant in an ANOVA comparing expression in treatment. 2 pairwise differences are significant: 380 vs. 2000 ppm (p=0.0113) and 750 vs 2000 ppm (p=0.00145). media type="custom" key="7803349"
 * December 9, 2010**

Vibrio exposure FAS expression by treatment. Differences in expression are not statistically significant. media type="custom" key="7804015"

__Ceramide: Primer optimization__ Sptlc1 and SMase Gradient qPCR (protocol on Friedman machine = ETS_gradient 52-59). Pooled control and VE samples from Vibrio exposure - 14 uL of each - to run one column of control and one of VE for each primer. media type="custom" key="7803913" Sptlc1 amplified very well (results [|link]). SMase will need qPCR protocol with more cycles, but also amplifies. Both primer sets yielded just one melt curve across annealing temps. 55C annealing temp should work fine with both primer sets, but higher temperatures (~58C) showed a narrower and stronger melt curve for SMase. media type="custom" key="7806435"

__NOAA OA: reverse transcription__ Reverse transcribed gDNA-free RNA samples (n=4). Put 2 ug of RNA (see table below "vibrio exposure") and brought volume up to 17.75 uL with H2O. Added 0.5 uL oligo dT primers and incubated at 70C for 5 min. Put on ice 5 min. Added 5 uL 5x MMLV buffer, 1.25 uL 10 mM dNTPs, and 0.5 uL reverse transcriptase (MMLV) to each sample. Incubated 42C for 1 hr + 95C for 3 min. Diluted samples (25uL) in 225 uL water and stored at -20 in Emma's cDNA Box III.
 * December 8, 2010**

__Vibrio exposure: reverse transcription__ Dnased RNA samples from SJW. Followed same procedure as above. "VE" samples (n=10) were exposed to live Vibrio vulnificus and V. parahaemolyticus for 24 hours at 2x10^11 CFU. There are also 10 controls, "C". SJW extracted RNA from gill tissues for all 20 animals on 2.15.2008 and Dnased 3.25.2008. media type="custom" key="7786203"

__NOAA OA: qPCR results__ [|Link] to results still evidence of contamination in 750E. There is also some contamination in a negative control, almost identical to the amount of gDNA contamination in the sample. Will rePCR all 3 samples this morning to check results. All samples were clean in qPCR (run jointly with Vt-challenge samples).
 * December 7, 2010**

__Vt-challenge qPCR__ Got samples of Vt-challenged adult C. gigas from SJW. Only a very small volume left in the samples, so decreased template volume to 0.5 uL per reaction. Did qPCR of 4 controls and 4 Vt-exposed oysters with FAS primers. media type="custom" key="7771265" qPCR results didn't look very good and Ct values were very variable (probably due to pipetting variability of 0.5 uL of template). But primers worked on samples. There is more RNA of these samples and I will reverse transcribe it tomorrow.

__Ceramide: sequencing__ Ran 1.5% agarose gel of sampled PCR'd 12.6.10 (loaded each well with full 25uL volume of PCR product). Used 5 uL of Bioline's Hyperladder I. Cut out bands marked with a green "*" for sequencing. (For Sptlc1, just cut out top band) media type="custom" key="7777623"

__Ceramide: Sequencing__ Prepped DNA and primer plates for sequencing of gel bands cut out 12.3.10.
 * December 6, 2010**

Table of sequence information for ceramide and nutrition pathway primer design media type="custom" key="7771079"

1291827761

cPCR based on qPCR protocol of tissue distribution samples to sequence qPCR products. thermal cycle program (QPCR55): 95C, 3min; 40x 95C 10s, 55C 10s, 72C 30s; 95C 10s. plate layout - G=gill cDNA, DG=digestive gland, Mu=muscle, Ma=mantle, - = negative control. MHC, SMase, Sptlc1, Lass5, NGF, FAS, IL1B, InsR, and Leptin refer to qPCR primer pairs. NOAA OA: qPCR to test for gDNA qPCR using 18s primers of samples DNased 12.3.10. Samples are on SJW's plate in column 5 and RNA is diluted 1:20 in water. Layout in col. 5 is:
 * MHC(G) || Lass5 - || InsR (G) ||
 * MHC - || NGF (mu) || InsR - ||
 * SMase (G) || NGF - || Leptin (G) ||
 * SMase (DG) || FAS (Ma) || Leptin - ||
 * Smase - || FAS (Mu) ||  ||
 * Sptlc1 (G) || FAS - ||  ||
 * Sptlc1 - || IL1B (G) ||  ||
 * Lass5 (Mu) || IL1B - ||  ||
 * A || 380A ||
 * B || 750E ||
 * C || 2000A ||
 * D || neg ||
 * E || neg ||
 * F || x ||
 * G || x ||
 * H || x ||

__NOAA OA, Cg Vt, & ceramide: qPCR__ OA - qPCR with 18s to test for gDNA contamination in RNA (see 12.2.10) Vt - juvenile gill tissue from Vt challenge 3.16.10. Test 4 control & 4 challenged with Hsp70 and Lass5 (and EF1a to normalize). Not done in duplicate. tissue distribution - test in duplicate of FAS, MHC_q, and Sptlc1. May have contaminated 2nd replicate of FAS gill with DG. media type="custom" key="7734193" Results ([|Link]): gDNA contamination in OA samples 380A, 750E, and 2000A. Will need to DNase. Hsp70 and Lass5 amplified well in the Vt-challenged juvenile gill tissue. FAS and MHC also amplified well, but Sptlc1 did not amplify. All melt curves look good except for 2nd replicate of FAS DG (well 8C), which will be excluded from all analyses.
 * December 3, 2010**

media type="custom" key="7736045" all pairwise comparisons between mantle and other tissues expression levels are significant (p=8.46e-4) media type="custom" key="7736061" all pairwise comparisons of expression levels in the tissues are significant. media type="custom" key="7759421" Hsp70 for C. gigas juvenile Vt-exposure: p=0.1908. media type="custom" key="7759445" Lass5 for C. gigas juvenile Vt-exposure: p=0.6139.

__NOAA OA: DNase__ Rigorous protocol for 380A, 750E and 2000A (see 11.6.09). For volumes see 12.2.10. Dnased samples stored in NOAA OA box at -80C.

__Ceramide: sequencing__ Gel bands to be sequenced media type="custom" key="7735793"

__Ceramide: tissue distribution qPCR__ Primers = Hsp70, complement receptor, complement C3, defensin media type="custom" key="7718531" Results [|link] only Hsp70 amplified (single melt curve). All NTCs were clean. media type="custom" key="7721663" ANOVA results: muscle-gill p=0.0066.
 * December 2, 2010**

__Ceramide: Gene discovery__ PCR for sequencing and prepped primer plate for sequencing. Only PCR'd select tissues that resulted in bands that were picked for sequencing last time. media type="custom" key="7723165" Primer plate has 7 uL of water and 3 uL of 10uM primer per well. Loaded entire PCR product onto 1.5% agarose gel. Row 1 is MHC_c gill through Lass 5 negative control. Row 2 is the remaining samples. 25uL of PCR product is in each well of row 1; row 2 wells were smaller so beginning with NGF negative control, 1/2 of each sample is in adjacent wells (tried to put all of NGF muscle in one well and it overflowed). Ran gel at 80V. Bands were cut out and stored at -20C. media type="custom" key="7725791"

__NOAA OA: RNA extractions__ Extracted RNA using TRI reagent (manufacturer's protocol) from 4 of the 12 samples taken Sept 14, 2010 at 48-hours post-fertilization in the different CO2 treatments. The samples extracted are C from 280ppm, A from 380 ppm, E from 750 ppm, and A from 2000 ppm. For homogenization step, thawed larvae were squished with pestle in 500 uL of TRI reagent 30 times then 500 uL more of TRI was added and samples were inverted a few times before incubation at room temp. All pellets were visible. RNA was resuspended in 50 uL of water and incubated at 55C for 5 min. Concentration was determined on Nanodrop in duplicate. RNA samples were stored at -80C in NOAA OA September 2010 box. 1 uL of each RNA sample was diluted in 19 uL of water for qPCR test of gDNA contamination (stored at -20 in cDNA box II). media type="custom" key="7724799"

__Ceramide: qPCR results & stats__ p-values are from Tukey's HSD based on aov fit model in R. Only significant values are reported. Error bars are standard deviations with a n=2 for each sample. Expression values are normalized to EF1a (each value is normalized to average within tissue EF1a, n=2). Leptin amplified in one replicate of the mantle tissue - C(t)=39.97. All genes had a single melt curve.
 * December 1, 2010**

Insulin Receptor media type="custom" key="7712813" mantle-dig.gland p=0.022 muscle-mantle p=0.044

Lass5 media type="custom" key="7712819" mantle-dig.gland p=0.00089 mantle-gill p=0.00253 muscle-gill p=0.000473 muscle-mantle p=0.0247

NGF media type="custom" key="7712831" mantle-gill p=0.0167

__Ceramide: qPCR__ Tissue distribution of genes: Lass5, NGF, Insulin receptor, Leptin, IL1B with EF1a as normalizing gene. (Samples from reverse transcription accomplished October 27, 2010.) media type="custom" key="7704471" All NTCs were clean and amplification was successful. Results [|link] media type="custom" key="7704485"
 * November 30, 2010**

__T/Vt: qPCR__ qPCR of samples A, B, D, E, F, G, H, & I for all 4 days (7.13-7.16). See below for details. PCR done on Friedman Lab machine using 3-step amp+melt. File: T-Vt EF1a 11.29.10. qPCR done in duplicate for all samples with 4 NTCs. Results [|link] Very little amplification of samples (only ~8 total, a couple per day). Redid qPCR (not in duplicate) but used 8 uL of cDNA template and decreased amount of water per reaction accordingly to 2.5 uL.
 * November 29, 2010**

Amplification plot for 8 uL qPCR with EF1a primers media type="custom" key="7683249" C(t) values for samples that successfully amplified in 8 uL template qPCR media type="custom" key="7683261" The samples in wells F are from chamber G, not E.

__T/Vt: DNasing__ Diluted samples for DNasing as described 11.19.10. Did TURBO's regular DNase protocol: 5 uL buffer + 1 uL enzyme in each sample and incubate at 37C for 30 min. Add 5 uL inactivation buffer and vortex 3x during 2 min incubation. Spin down at 10,000 xg for 1.5 min, transfer supernatant to new tube and store at -80C (in Sarah & Emma's OA/Vt box). qPCR of DNased samples to test for gDNA contamination. Diluted 1 uL of DNased RNA in 19 uL of water to use as template. Each reaction had 12.5 uL Immomix, 0.5 uL of each primer, 1 uL of 50 uM SYTO13, 6.5 uL water, and 4 uL of template. Loaded samples in following order: No amplification of any of the samples so can make cDNA. Results [|link].
 * November 22, 2010**
 * 7.14A ||
 * 7.14D ||
 * 7.14F ||
 * 7.14H ||
 * 7.15E ||
 * 7.15G ||
 * neg ||
 * neg ||

__Reverse Transcription__ RT reactions are based on 2 ug of RNA. The following table outlines how much volume should be used for each sample to equate 2 ug. If a vol > 17.75 uL is required, the maximum of 17.75 uL is used for that reaction (for an amount <2ug). Samples diluted in 225 uL of water and stored at -20 in Emma's cDNA Box III.

__T/Vt: Sample prep__ qPCR of T/Vt samples taken 7.13, 7.14, and 7.15.10 (previously analyzed samples from 7.16.10). Used 18s primers and amplified diluted RNA (1:20) to test for gDNA contamination. Results [|link] media type="custom" key="7592973" Some samples had gDNA contamination. Will Dnase Monday - details for DNasing below. media type="custom" key="7593321"
 * November 19, 2010**

Found concentrations for all samples on Nanodrop (concentrations taken in duplicate then averaged). The chart below is of RNA concentration and 260/280. If 260/280=2.1, the RNA is pure. Anything between 1.8 and 2.1 is considered good quality RNA. media type="custom" key="7593229" media type="custom" key="7593233"

__Ceramide: Gel of PCR 11.12.10__ Made 200 mL 1.5% agarose gel (200 mL 1xTAE + 3 g agarose + 20 uL EtBr). Loaded 10 uL of PCR product in each well (5 uL hyperladder). Ran at 80V for 45 minutes. Cut out bands indicated below and put in cDNA Box II (-20C). Bands are indicated with white arrows and labeled with band name that corresponds to storage tube. Accidentally loaded loading buffer instead of hyperladder on gel, but can still get rough idea of most of the PCR products being the correct size (<200 bp). media type="custom" key="7532063"
 * November 13, 2010**

Ceramide Received primers for ceramide pathway. Eluted to 100 uM with TE buffer (pH 8.5).
 * November 12, 2010**


 * Primer || SRID ||  ||
 * FAS F || 1019 ||  ||
 * FAS R || 1020 ||  ||
 * NGF F || 1021 ||  ||
 * NGF R || 1022 ||  ||
 * IL1B F || 1023 ||  ||
 * IL1B R || 1024 ||  ||
 * Lass5 F || 1025 ||  ||
 * Lass5 R || 1026 ||  ||

PCR of cDNA (11.11.2010) with new primers. Diluted cDNA in 225uL water; It looked like T.ISO 3 hemolymph sample had evaporated a little. Each reaction has 12.5 uL Apex master mix, 8.5 uL water, and 0.5 uL of each primer (10 uM). 22 uL of master mix and 3 uL of cDNA template in each well. PCR55 program on thermocycler (generic, 55C annealing). Plate layout below. media type="custom" key="7517129"

__**November 11, 2010**__ __Ceramide/Nutrition: Dnasing__ Diluted samples according to volumes for 50 uL (11/8/10). Dnased all samples according to Ambion's TURBO protocol. For both TISO3 and PLY3, followed rigorous protocol for mantle, gill & muscle and followed regular protocol for visceral mass & hemolymph. Regular: added 5 uL buffer + 1 uL Dnase and incubated 30 min at 37C. For rigorous: added 5 uL buffer + 0.5 uL Dnase, incubated 37C for 30 min then added 0.5 uL Dnase and incubated again. At end of incubations added 5 uL inactivation buffer and vortexed 3x over 2 min. Centrifuged at 10000xg for 1.5 minutes and put supernatant in new tube (stored at -80).

qPCR with 18s primers of Dnased RNA to test for gDNA contamination. media type="custom" key="7503985" media type="custom" key="7503979" Results [|link]

__Reverse transcription__ Reverse transcribed Dnased RNA to cDNA following protocol from November 6, 2009. Used random primers instead of Oligo dTs.

__**November 10, 2010**__ __Environmental monitoring__ Retrieved PSDs (2) from the Aquarium at 10 am. Water level was higher than when they had been deployed and there were many jellies near the surface. There was a shrimp living in the "anchor" (cement block). Put each PSD in its own ziplock back and transported back to UW on ice (now stored in -20 freezer). Attachments on long-line hooks had corroded so would need to find a different way of attaching the hooks to the rope for longer deployment. The hooks are supposed to corrode so that they detach from lost/abandoned gear.

__**November 8, 2010**__ __Ceramide/Nutrition: RNA extractions__ Extracted RNA from tissues collected oct. 29, 2010 using TRI reagent. Tissues extracted are for 1 oyster from each treatment (PLY3 & T.ISO 3) and include gill, hemolymph*, visceral mass, mantle & muscle. Resolubilized RNA pellets (visible pellets in all samples) in water: 50 uL for visceral mass, muscle & hemolymph; 100 uL for gill & mantle. Measured RNA concentrations in triplicate on Nanodrop. Stored samples at -80 in NOAA OA larvae box. media type="custom" key="7463033"


 * note of concern: do the hemolymph samples contain gametes?

__**November 2, 2010**__ __Ceramide pathway: Sequencing__ Table of expected sequence sizes vs. sizes of amplified PCR product from gels 10.27.10 media type="custom" key="7398153" Fragments in pink boxes are those that will be sequenced. Bands to be sequenced with just forward primer: 1-15, 2-3b, 2-10, 1-1b, 1-11a, 3-1b, 3-1c, 3-8 Bands to be sequenced with both primers (>600 bp): 2-3a, 2-4, 2-6, 1-1a, 1-2b, 3-1a

Purified bands in Millipore columns - put bands in gel nebulizer of column and spun down at 5,000xg for 10 min. Put 10 uL of sample in appropriate well (see DNA plate layout below) except for band 1-2b (7 uL purified band + 3 uL water). For primers, put 3 uL of F or R primer + 7 uL water in each well (see below). Gave plates to SJW for sequencing. media type="custom" key="7399229"

__**October 29, 2010**__ __Nutrition__ Terminated algae nutrition experiment. Sampled 3 oysters from each treatment (T.iso and Ply). Took 5 different tissue samples for the 6 oysters: gill, hemolymph, mantle, muscle, digestive gland/visceral mass/gonad. Most of the oysters had developed gonad, which was difficult to distinguish from the visceral mass/DG. Tissues were stored in 1 mL RNAlater at 4C except for hemolymph which was stored in 0.5 mL TRI reagent at 4C. Shucker was cleaned between oysters and dissecting tools were cleaned between tissues in bleach & EtOH.

__**October 27, 2010**__ __Ceramide pathway__ Reverse transcription of DNased RNA samples. Put 16.75 uL water, 1 uL RNA, and 1 uL Oligo dT primers (oops, should have been 0.5 uL) into wells of a plate. Heated at 70C for 5 minutes and put on ice for ~3 minutes. Added 5 uL MMLV buffer, 1.25 uL 10 mM dNTPs and 0.5 uL reverse transcriptase. Heated at 42C 1 hours, 95C 3 min.

Diluted cDNA in 225 uL water. Stored at -20 in cDNA box II.

Primer test with cPCR: test of all new primers (qPCR: leptin, insulin receptor, SMase, Sptlc1, MHC; cPCR: SMase, MHC). For each primer pair, amplified cDNA from gill, mantle, muscle, dig. gland, and hemolymph + 1 negative control. Used Apex master mix: 12.5 uL Apex, 8.5 uL water, 0.5 uL each primer. Thermal profile: 95C 10 min; 95C 30s, 55C 30s, 72C 30s (repeat 40x); 72C 10 min. media type="custom" key="7334545"

Made 1.5% agarose gel (100 mL 1xTAE, 1.5 g agarose, 10 uL EtBr). Loaded gel with hyperladder I (Bioline, see below) in the beginning of both rows (5 uL). Row 1 has samples MHC (c) gill through MHC (q) muscle. Row 2 has samples MHC (q) DG through Sptlc1 negative control. Ladder media type="custom" key="7336137" __Gel1__ media type="custom" key="7335827" Close-up of MHC (c) media type="custom" key="7335833" Made 50 mL 1.5% agarose gel to run remaining samples. First row on Gel 2 (Row 3) was InsReceptor Gill through Leptin DG. Row 4 was Leptin hemolymph, positive, and negative controls. media type="custom" key="7335885" Letters at the heads of gel lanes correspond to tissue type from which cDNA was derived: G=gill, Ma=mantle, Mu=muscle,DG=digestive gland, H=hemolymph, +=positive control (Cg control 3 from cDNA box II), - = negative control. Bands that are labeled (i.e. 1-1c) were cut out for sequencing. Bands are in eppie tubes at -20C. Possible contamination in negative controls for MHC (c) and SMase (c). SMase may be primer-dimer.

__Algae counts & feeding: Nutrition__ Counted algae and updated spreadsheet. Fed 17.5 mL T.ISO (~165000 cells/mL) and 100 mL PLY (175000 cells/mL).

__**October 26, 2010**__ __Algae counts & feeding: Nutrition__ Counted algae from 10.1-10.12 flasks (300 mL). Fed 15 mL of T.ISO (185,000 cells/mL) and 125 mL/remaining in flask of PLY (200000 cells/mL).

__Ceramide pathway__ qPCR to test gDNA contamination of RNA extracted 10.20 and to test new primers: leptin, insulin receptor, MHC, sphingomyelinase, sptlc1. All primers were reconstituted today to 100 uM with Tris HCl buffer (pH 8.5). Locations in Primer Box #7 and SRID are as follows:

F2 Cg_MHC_qPCR_F 996 C.gigas 10/26/2010 F3 Cg_MHC_qPCR_R 995 C.gigas 10/26/2010 F4 Cg_SMase-qPCR_F 994 C.gigas 10/26/2010 F5 Cg_SMase-qPCR_R 993 C.gigas 10/26/2010 F6 Cg_Sptlc1-qPCR_F 992 C.gigas 10/26/2010 F7 Cg_Sptlc1-qPCR_R 991 C.gigas 10/26/2010 F8 Cg_MHC_cPCR_F 990 C.gigas 10/26/2010 F9 Cg_MHC_cPCR_R 989 C.gigas 10/26/2010 G1 Cg_SMase-cPCR_F 987 C.gigas 10/26/2010 G2 Cg_SMase-cPCR_R 986 C.gigas 10/26/2010 G3 CgInsulinR-F 985 C.gigas 10/26/2010 G4 CgInsulinR-R 984 C.gigas 10/26/2010 G5 CgLeptin-F 983 C.gigas 10/26/2010 G6 CgLeptin-R 982 C.gigas 10/26/2010

qPCR on DNased RNA (see 10.25.10) was done with 18s primers with duplicates of samples. RNA was diluted 1:20 in water before added to the reaction. qPCR to test primers insulin, leptin, MHC, SMase, and Sptlc1 was done on cDNA from OADev (samples 380 B1 and 840 A1, in duplicate). media type="custom" key="7330841" Results: All new qPCR primers worked in at least one sample except for SMase. There was no amplification of the RNA samples, meaning there is no evidence of gDNA contamination and samples can be reverse transcribed. The gDNA control amplified with the 18s primers. There was no evidence of contamination in any of the NTCs. media type="custom" key="7330781" media type="custom" key="7330787"

__**October 25, 2010**__ __Ceramide pathway__ Used nanodrop to find RNA concentrations from extractions 10.20.10. Took concentrations in triplicate of each sample - gill, dig. gland, mantle, muscle, hemolymph. Calculated number of uL needed for 10 ug of RNA and brought up to 50 uL with water. Hemolymph was the only sample that required a volume >50uL for 10 ug so used entire sample. media type="custom" key="7307283" DNased RNA samples, did rigorous protocol for all except did regular for hemolymph since RNA concentration was so low. See March 18, 2010 for details on rigorous protocol. For rigorous added 0.5 uL DNase + 5 uL 10x buffer to samples and incubated at 37C for 30 min; added additional 0.5 uL DNase and incubated another 30 minutes. Regular protocol: added 1 uL DNase + 5 uL buffer and incubated at 37 C for 30 min. After incubations, added 5 uL inactivation buffer and vortexed 3 x over 2 minutes. Spun at 10,000xg for 1.5 min and put supernatant in new tube. Stored at -80.

__Algae counts & feeding: Nutrition__ Karissa counted algae from 10.19 flasks (300 mL). Fed 30 mL of T.ISO (210000 cells/mL) and 90 mL of PLY (171000 cells/mL). Made 2L new media from water Zac autoclaved last week. Seeded new flasks of PLY and T.ISO.

__Algae counts & feeding: Nutrition__ Counted algae from feeder flask for T.ISO and from 300 mL 10.19 for PLY. Significant numbers of dead algae cells (most were still alive) in T.ISO so after feeding discarded remaining algae. Fed ~360,000 cells /mL of T.ISO and 340,000 cells/mL of PLY (T.ISO probably an overestimate given number of dead algal cells in culture).
 * October 24, 2010**

__Algae counts & feeding: Nutrition__ Counted algae - did not add any EtOH. Updated counts are in spreadsheet. Fed oysters ~11 mL of T.ISO (170000 cells/mL) and the rest of the PLY (~40 mL, 196000 cells/mL). Before feeding changed water in tanks.
 * October 22, 2010**

__Algae counts & feeding: Nutrition__ Counted algae. Added 50 uL of 75% EtOH to 1 mL of algae from feeding flasks. Still not enough EtOH to completely stop swimming, but algae were slower. Oysters were fed 15 mL T.ISO and 77 mL PLY.
 * October 21, 2010**

__Ceramide pathway__ Extracted RNA from tissues collected yesterday using TRI reagent. All pellets were resuspended in 100 uL water, except hemolymph was resuspended in 50 uL. Resuspension was aided by incubation at 50C for ~5 minutes, samples were then lightly vortexed and stored at -80 in NOAA Sept 2010 box.
 * October 20, 2010**

__Algae counts & feeding: Nutrition__ Counted algae from 300 mL flasks 10/1-10/12 because did not see other flasks which had been taken out of the hood. Added 10 uL 75% EtOH to 1 mL of algae to slow algae movement. Took 2 aliquots from the 1 mL and counted 6 squares on hemacytometer. This did not have much of an effect on the algae. Oysters were fed 20 mL T.ISO and 155 mL PLY, ~170000 cells/mL each. Fed the trash can oysters 10 mL SD1800.

__Biomonitoring__ Deployed 2 PSDs at the Aquarium (POCIS). PSDs were attached to a rope using long-line clips. The rope was weighted with a large cinder block (PSDs were ~5-6' above the block). The block & PSDs were lowered into the water until the block touched bottom and then the top of the rope was tied off to secure vertical suspension of the PSDs in the water column. PSDs will be deployed for 3 weeks.

__Algae counts & feeding: Nutrition__ Counted algae (see 10.13) and updated spread sheet. Fed oysters 14 mL T.ISO and 47 mL PLY (~170000 cells/mL each). White/grayish debris in bottom of tanks is probably feces - looked under 'scope and they were dense and solid. Also under scope found larvae from the tanks - surprise barnacle larvae!!
 * October 19,2010**

Cleaned out tanks by emptying water, rinsing twice with dechlorinated water and refilling with new seawater until oysters covered. Fed oysters after cleaned tanks.

__Ceramide pathway__ Dissected oyster from yesterday. Tissues for RNA extraction (gill, mantle, muscle, digestive gland, and hemolymph) were put in 0.5 mL RNAlater at 4C; duplicates of all tissues except hemolymph were frozen immediately at -80C. The frozen tissues are in the C. gigas Larvae NOAA OA September 2010 box.

__Algae counts & feeding: Nutrition__ Counted algae (see 10.13) and updated spread sheet. Fed oysters 20 mL TISO and 142 mL PLY (2x normal feeding because missed feeding this weekend, ~340,000 cells/mL). Oysters were all sightly agape. Weird white stuff on bottom of PLY treatment tank - will investigate tomorrow. Will also change water in both tanks tomorrow.
 * October 18, 2010**

__Environmental monitoring__ Picked up 2 PSDs from Irv Shultz (PNNL). Irv has membranes mounted inside steamers (cooking appliance) and held closed with zip ties. This would save $355 per site ($1065 total).

__Ceramide signaling pathway__ Picked up an oyster from Taylor shellfish to dissect for various tissues and test ceramide pathway primers. Put oyster in tank with saltwater.

**October 15, 2010** __Algae counts & feeding: Nutrition__ Counted algae as outlined 10.13.10 and updated spread sheet. Fed oysters 15 mL T.ISO and 95 mL PLY (~1.7*10^5 cells/mL). Oysters in both treatments were slightly agape. Fed trashcan oysters 10 mL SD1800. Oysters will not be fed 10.16.10.

__Algae counts & feeding: Nutrition__ Counted algae as outlined 10.13.10 and updated spread sheet. There was more cells/uL of T.ISO today but not of PLY. Fed the oysters 25 mL of T.ISO (1.66x10^5 cells/mL) and 115 mL of PLY (same # cells/mL). Oysters in T.ISO treatment are mostly closed and barnacles are inactive. This is in stark contrast to the oysters in the PLY treatment where all barnacles are active and all oysters are slightly agape. Fed trashcan oysters 10 mL SD1800. Received leptin pathway primers (2 pairs) but did not elute.
 * October 14, 2010**

**October 13, 2010** __Algae counts & set-up: Nutrition__ Swirled algae flasks so well-mixed. Took aliquots from both algaes, Isochrysis (T. ISO) and tetraselmis (PLY). Counted 2 ~15 uL aliquots from each algae type on hemacytometer. Counts were for number of algae cells in 6 different squares as defined by the pink square (below). T.ISO cells were much smaller than PLY cells. Ongoing algae counts can be found in spreadsheet [|here]. media type="custom" key="7185447" Put oysters (collected at Big Beef Creek) in 2 separate rectangular experimental tanks (n=4 for T.ISO and n=5 for PLY). Each tank has a small pump to create water flow. Fed the PLY treatment 100 mL of algae and the T.ISO treatment got 25 mL of algae. Oysters in trash can were fed 10 mL of shellfish diet 1800.

**October 11, 2010** __ Primer design: Ceramide Signalling Pathway __ Designed primers for sequencing and qPCR for genes in the ceramide pathway. Known vertebrate sequences for the genes and their C. gigas homologues were aligned in Geneious. In some cases (MHC, Lass5, and SMase) consensus sequences for C. gigas were created based on multiple overlapping ESTs. For MHC and SMase, there is not a published EST for the entire ORF so primers were designed based on the consensus sequences to include the entire ORF. QPCR primers were designed for MHC, SMase, and Sptlc1. media type="custom" key="7185525" yellow arrows designate open reading frames in the oyster sequence (1) and the Bombyx mori homologue (2).

**October 7, 2010** __Bioinformatics: Ceramide Signalling Pathway__ Read more papers on ceramide (see My Delicious, "ceramide" bookmark). Got NCBI sequences from top BLAST hits (10.6.10). Blasted these top hits against NCBI nucleotide and est databases for cross-reference purposes. Will have to determine e-value cut-off for determining if gene is really in oyster ceramide pathway. Sequences will be the basis for primer design.

Bioinformatics: Ceramide Signalling Pathway__ Compiled list of genes in the pathway based on Ballou et al. 1996: Ceramide signalling and the immune response. Reference sequences in organisms with the genes in the pathway that are published on NCBI were compiled (see [|spreadsheet]). For each gene, sequences were entered into blastall (wet genes) with the following settings: tblastx (nucleotide query transl./transl. nuc. db); protein db "nr"; nucleotide db "cgigas...Sigenae"; tabulated output. (All other settings default).
 * October 6, 2010**