Information on diagnostic assays. Chronological order, each entry started with date. contact info: sroberts@mbl.edu 508-289-7621 sequences of primers / probes used listed at top of page (5'-3') NVtmf TCG CTG GAG TGT ACG TCT CAG T NVtmr GAG TGG TCC GAG GGT TAG GAT NVcap fam- CAT CCC TTG AGA CGC CCG AAC -bhQ1 Funiv TCT TCC AGC GAT ACG CTG TTG A Runiv TCA GTG TTG TTG CCG RCA CAC A ps7L CCA AGA TTG GGA TAC GCT GCT AGA ps7R GTG TTC CCG TAT TCC CAG CTA CTT RNA1F TCG TCA AGG CGT TCC AAA AG RNA1R GAC AGA CAT CTT CGG TTC CAA ATC F1 CCA AAT GGT GGG AAA GCA GAA C R1 GTT GTA GAG TGG TCC GAG GGT TAG _______________________________________________________________________________________ 10/4/2003 samples 1-8 removed from RNAlater - extracted for total RNA using TriReagent in 50ul reaction volumes samples were assayed under following conditions sense primer (NVtmf) 0.2 ul - 80 mM (final conc.) antisense primer (NVtmr) 0.2 ul - 80 mM fluorogenic probe (NVcap) 0.5 ul - 200 mM Dual enzyme mix 1.0 ul MgSo4 1.0 ul - 5mM 2x reaction mix - 25 ul Condtions (Standard) 50 degrees 30 min 95 d 5 min 95 d 15 sec 60 d 1 min 40 cycles - detecting fluoresence every cycle. samples run at .1 ug and .6 ug no samples (1-8) were positive under these conditions positive control samples were positve - (cDNA preps) negative control samples were negative - (water) _________________________________________________________________________ 10/5/2003 Samples 1-8 assayed using Stratagene SYBR green RT_PCR system Reaction volumes water 21.5 ul 2x mix 25 ul F primer (Funiv) 0.2 ul R primer (Runiv) 0.2 ul RTRNase BLock 0.125 ul Template 3 ul (o.6 ug total) and Reaction volumes water 21.5 ul 2x mix 25 ul F primer (ps7L) 0.2 ul R primer (ps7R) 0.2 ul RTRNase BLock 0.125 ul Template 3 ul (o.6 ug total) Protocol Setup 1. Incubate at 50.0 C for 00:30:00 2. Incubate at 95.0 C for 00:10:00 3. Incubate at 95.0 C for 00:00:30 4. Incubate at 63.0 C for 00:01:00 5. Plate Read 6. Incubate at 72.0 C for 00:00:30 7. Plate Read 8. Goto line 3 for 39 more times 9. Incubate at 95.0 C for 00:01:00 10. Incubate at 55.0 C for 00:00:01 Manual ramp rate of 0.2 C per second 11. Melting Curve from 55.0 C to 95.0 C 12. Incubate at 21.0 C forever END *POSTIVE control samples did not work -- therefore no results generated. ________________________________________________________________________ 10/6/2003 Samples 1-8 assayed using Stratagene SYBR green RT_PCR system Reaction volumes water 21.5 ul 2x mix 25 ul F primer (Funiv) 0.2 ul R primer (Runiv) 0.2 ul RTRNase BLock 0.125 ul Template 3 ul (o.6 ug total) Protocol Setup 1. Incubate at 50.0 C for 00:30:00 2. Incubate at 95.0 C for 00:10:00 3. Incubate at 95.0 C for 00:00:30 4. Incubate at 50.0 C for 00:01:00 *NOTE dropped temperature 5. Plate Read 6. Incubate at 72.0 C for 00:00:30 7. Plate Read 8. Goto line 3 for 39 more times 9. Incubate at 95.0 C for 00:01:00 10. Incubate at 55.0 C for 00:00:01 Manual ramp rate of 0.2 C per second 11. Melting Curve from 55.0 C to 95.0 C 12. Incubate at 21.0 C forever END *POSTIVE control samples did not work -- therefore still no results generated. __________________________________________________________________________ 10/7/2003 Samples 1-8 were re-extracted for RNA as only 1/2 was used in initial extraction. RNA was Reverse Transcribed Reagents 5 ul RNA 75 degrees 5min ice add to Mix AMV Buffer 4ul dNTP 2.5 mM 8 ul AMV RT 1ul Random primer 1ul h20 1ul 21 degrees 10min 37 degrees 60 min 95 degrees 3 min RT product PCR'd using Amplitaq (Perkin Elmer) Amplitaq Buffer 5ul dNTP 4ul Taq 0.2 ul F primer 5 ul **multiple primer combos RNA1f, F1, ps7L R primer 5 ul RNA1r, R1, ps7R h20 28.8 ul 95 95 55 72 40 cycles 72 *All positive CONTROL samples were positive with all 3 primer pairs. All of the unknown samples (1-8) were negative. ___________________________________________________________________________ 10/22/03 Cd samples were extracted for RNA 10 samples no tag no tag a no tag d 0142 0128 0122 0036 0145 0115 in 50ul reaction volumes samples were assayed under following conditions sense primer (NVtmf) 0.2 ul - 80 mM (final conc.) antisense primer (NVtmr) 0.2 ul - 80 mM fluorogenic probe (NVcap) 0.5 ul - 200 mM Dual enzyme mix 1.0 ul MgSo4 1.0 ul - 5mM 2x reaction mix - 25 ul Condtions (Standard) 50 degrees 30 min 95 d 5 min 95 d 15 sec 60 d 1 min 40 cycles - detecting fluoresence every cycle. samples run at .3 ug no samples (1-10) were positive under these conditions positive control samples were positve - (cDNA preps) negative control samples were negative - (water) ___________________________________________________________ 10/23/04 microtec samples (.01g tiss) extracted for RNA in 50ul reaction volumes samples were assayed under following conditions sense primer (NVtmf) 0.2 ul - 80 mM (final conc.) antisense primer (NVtmr) 0.2 ul - 80 mM fluorogenic probe (NVcap) 0.5 ul - 200 mM Dual enzyme mix 1.0 ul MgSo4 1.0 ul - 5mM 2x reaction mix - 25 ul Condtions (Standard) 50 degrees 30 min 95 d 5 min 95 d 15 sec 60 d 1 min 40 cycles - detecting fluoresence every cycle. samples run at .3 ug no samples were positive under these conditions positive control samples were positve - (cDNA preps) negative control samples were negative - (water) _____________________________________________________________ 10/24/03 4 microtech samples termed 'pos' Reagents 5 ul RNA 75 degrees 5min ice add to Mix AMV Buffer 4ul dNTP 2.5 mM 8 ul AMV RT 1ul Random primer 1ul h20 1ul 21 degrees 10min 37 degrees 60 min 95 degrees 3 min RT product PCR'd using Amplitaq (Perkin Elmer) Amplitaq Buffer 5ul dNTP 4ul Taq 0.2 ul F primer 5 ul **multiple primer combos RNA1f, F1, ps7L R primer 5 ul RNA1r, R1, ps7R h20 28.8 ul 95 95 55 72 40 cycles 72 *All positive CONTROL samples were positive with all 3 primer pairs. All of the samples (1-4) were negative. __________________________________________________________________ 10/30/03 Extracted .1 ug tissue form Hdk (6 samples) 575 92 96 56 20567 91 Ran SYBR green (2 primer pairs) 25 ul rxn Water 11.2 2x Mix 12.5 F primer 0.125 *Funiv, RNA1 F R primer 0.125 Runiv, RNA1 R RT/RNASe block 0.0625 Cycling as indicated 1. Incubate at 50.0 C for 00:30:00 2. Incubate at 95.0 C for 00:10:00 3. Incubate at 95.0 C for 00:00:30 4. Incubate at 50.0 C for 00:01:00 5. Plate Read 6. Incubate at 72.0 C for 00:00:30 7. Plate Read 8. Goto line 3 for 39 more times 9. Incubate at 95.0 C for 00:01:00 10. Incubate at 55.0 C for 00:00:01 Manual ramp rate of 0.2 C per second 11. Melting Curve from 55.0 C to 95.0 C read every 0.5 C hold 00:00:30 12. Incubate at 21.0 C forever END Univ primers did not work even with positive control. Will discontinue using this primer pair RNA1 primers - positive control was pos, all unknowns were negative. ____________________________________________________________________________________ 10/31/03 Extracted Pomp (3) A1 A2 A3 Extracted Gpr (4) H2 H4 H9 H10 Ran SYBR green (2 primer pairs) On above samples and 6 Hdk from 10/30 25 ul rxn Water 11.2 2x Mix 12.5 F primer 0.125 *Ps7L, F1 R primer 0.125 Ps7R, R1 RT/RNASe block 0.0625 Protocol Setup Temperature Control: Sample Calculation Lid Mode: Constant 100.0C; Shutoff < 30.0C 1. Incubate at 50.0 C for 00:30:00 2. Incubate at 95.0 C for 00:10:00 3. Incubate at 95.0 C for 00:00:30 4. Incubate at 50.0 C for 00:01:00 5. Incubate at 72.0 C for 00:00:30 6. Plate Read 7. Goto line 3 for 39 more times 8. Incubate at 95.0 C for 00:01:00 9. Incubate at 55.0 C for 00:00:01 Manual ramp rate of 0.2 C per second 10. Melting Curve from 55.0 C to 95.0 C read every 0.5 C hold 00:00:30 11. Incubate at 21.0 C forever END ps7 primer pair A1 and A2 potential F1/R1 primer pair multiple potential on Hdk samples and 1 Gpr ______________________________________________________________________________________ 01/15/04 Extracted fB 1-04 mC 1-04 E4 E5 f 1-04 E3 mB 1-04 mA 1-04 E12 Ran SYBR green (2 primer pairs) On above samples 25 ul rxn Water 6.2 2x Mix 12.5 F primer 0.125 *Ps7L, F1 R primer 0.125 Ps7R, R1 RT/RNASe block 0.0625 template 6 ul (0.6 ug total) Protocol Setup Temperature Control: Sample Calculation Lid Mode: Constant 100.0C; Shutoff < 30.0C 1. Incubate at 50.0 C for 00:30:00 2. Incubate at 95.0 C for 00:10:00 3. Incubate at 95.0 C for 00:00:30 4. Incubate at 50.0 C for 00:01:00 5. Incubate at 72.0 C for 00:00:30 6. Plate Read 7. Goto line 3 for 39 more times 8. Incubate at 95.0 C for 00:01:00 9. Incubate at 55.0 C for 00:00:01 Manual ramp rate of 0.2 C per second 10. Melting Curve from 55.0 C to 95.0 C read every 0.5 C hold 00:00:30 11. Incubate at 21.0 C forever END All samples were negative Positive RNA control run for both primer pairs tested positive. Negative controls (no template) tested negative. --------------------------------------------------------------------------------------- 9/13/04 Samples analyzed with Ps7L/Ps7R and NodaF1/NodaR1 using SYBR Green RT-PCR system under the following conditions: Water 6.2 ul 2X Brilliant Green Master Mix 12.5 ul 10 uM Primer fwd 0.125 ul 10 uM Primer rev 0.125 ul RT RNase Block 0.0625 ul 0.2 ug/ul RNA 6 ul Reaction volume 25 ul ÒNoda1Ó: 1. 50oC Ð 30 minutes 2. 95oC Ð 10 minutes 3. 95oC Ð 30 seconds 4. 50oC Ð 1 minutes 5. 72oC Ð 30 minutes 6. Plate Read 7. GoTo Step 3 39 more times 8. 95oC Ð 1 minutes 9. 55oC Ð 1 second; ramp 0.2oC/second 10. Melting Curve from 55-95oC, read every 0.5 oC, hold 30 seconds 11. Hold 21oC forever 12. End Results: Amplification in positive controls. No amplification in negative controls. No amplification in samples. Samples Included: #1486 Brain 1/5/04 Brain 10/30/03 Brain 10/31/03 Brain 10/31/03 Gonad 1/5/04 Gonad #1486 Ovary 3/20/03 gbFingerling Ô1Õ 3/20/03 gbFingerling Ô2Õ 3/20/03 gbFingerling Ô3Õ 3/28 Brain Jumper 12/3/03 150D*2 Brain 12/14/03 6 Eggs 12/14/03 3 Milt water 12/14/03 4 Milt 12/14/03 7 Milt 10/30/03 Eggs 12/27/03 Eggs gbfish #1486 Blood ------------------------------------------------------------------------------------------ 9/15/04 Samples analyzed with NVtmf/tmR/cap (dual label probe RT PCR) as follows: Water 16.1 ul 2X Reaction Mix 25 ul 10 uM NVtmf 0.2 ul 10 uM NVtmr 0.2 ul 10 uM Nvcap (probe) 0.5 ul Dual Enzyme Mix 1 ul 0.2 ug/ul RNA 6 ul* Reaction volume 50 ul *1.5 ul of each positive control template was used ?one step 60?: 1. 50oC ? 30 minutes 2. 95oC ? 5 minutes 3. 95oC ? 15 seconds 4. 60oC ? 1 minute 5. Plate Read 6. GoTo Step 3, 39 more times 7. 21oC ? forever 8. End Results: Positive controls did not work. Negative controls (2 of 3) displayed fluorescence. No results generated. --------------------------------------------------------------------------------------------------- 10/05/2004 Extracted and quantified RNA from Haddock Larvae Ozone Egg Treatment 5/10/04 ? S. Lindell Label weight (g) 1X Tri (ml) date extract vol RNA (ul) dil A260 ug/ul A1 ~.035 0.5 10/5/2004 20 1:99 0.152 0.608 A2 ~.035 0.5 10/5/2004 20 1:99 0.154 0.616 A3 ~.035 0.5 10/5/2004 20 1:99 0.279 1.116 1A1 ~.035 0.5 10/5/2004 20 1:99 0.116 0.464 1A2 ~.035 0.5 10/5/2004 20 1:99 0.282 1.128 1A3 ~.035 0.5 10/5/2004 20 1:99 0.561 2.244 B1 ~.035 0.5 10/5/2004 20 1:99 0.127 0.508 B2 ~.035 0.5 10/5/2004 20 1:99 0.27 1.08 B3 ~.035 0.5 10/5/2004 20 1:99 0.11 0.44 1B1 ~.035 0.5 10/5/2004 20 1:99 0.57 2.28 1B2 ~.035 0.5 10/5/2004 20 1:99 0.287 1.148 1B3 ~.035 0.5 10/5/2004 20 1:99 0.11 0.44 ------------------------------------------------------------------------------------------------- 10/06/2004 Extracted and quantified RNA from Haddock Larvae Ozone Egg Treatment 5/10/04 ? S. Lindell Label weight (g) 1X Tri (ml) date extract vol RNA (ul) dil A260 ug/ul 2A1 ~.035 0.5 10/6/2004 20 1:99 0.147 0.588 2A2 ~.035 0.5 10/6/2004 10 1:99 0.179 0.716 2A3 ~.035 0.5 10/6/2004 20 1:99 0.169 0.676 2B1 ~.035 0.5 10/6/2004 20 1:99 0.051 0.204 2B2 ~.035 0.5 10/6/2004 20 1:99 0.206 0.824 2B3 ~.035 0.5 10/6/2004 20 1:99 0.152 0.608 4A1 ~.035 0.5 10/6/2004 20 1:99 0.057 0.228 4A2 ~.035 0.5 10/6/2004 20 1:99 0.259 1.036 4A3 ~.035 0.5 10/6/2004 20 1:99 0.118 0.472 4B1 ~.035 0.5 10/6/2004 10 1:99 0.182 0.728 4B2 ~.035 0.5 10/6/2004 20 1:99 0.059 0.236 4B3 ~.035 0.5 10/6/2004 20 1:99 0.165 0.66 ---------------------------------------------------------------------------------------------------- 10/7/2004 The following samples were analyzed with NVtmf/tmR/cap (dual label probe RT PCR) as follows: #1486 Brain 3/20/03 gb Fingerling 3 Water 16.1 ul 1/5/04 cod brain 3/28 COD Brain Jumper 2X Reaction Mix 25 ul #1486 Brain 12/3/03 Brain 150D*2 COD 10 uM NVtmf 0.2 ul 1/5/04 cod brain 12/14/03 6 Eggs 10 uM NVtmr 0.2 ul 10/30/03 cod brain 12/14/03 3 milt 10 uM Nvcap (probe) 0.5 ul 10/31/03 cod brain 12/14/03 4 milt Dual Enzyme Mix 1 ul 10/31/04 cod gonad 12/14/03 7 milt 0.2 ug/ul RNA 10 ul* 1/5/04 cod gonad 10/30/03 COD Eggs Reaction volume 50 ul #1486 ovary 12/27/03 COD Eggs GBA fish 3/20/03 gb Fingerling 1 #1486 Blood 3/20/03 gb Fingerling 2 *4 ul of each positive control template and 6 ul water were used ?one step 60?: 1. 50oC ? 30 minutes 2. 95oC ? 5 minutes 3. 95oC ? 15 seconds 4. 60oC ? 1 minute 5. Plate Read 6. GoTo Step 3, 39 more times 7. 21oC ? forever 8. End Results: No amplification in Negative Template Controls. Positive Control Templates amplified with early C(t) values (10 and 12 cycles). All samples were negative. One sample, #1486 Blood, displayed a different fluorescence pattern than the other samples. This may indicate that the RNA extraction was not successful and may need a different method due the nature of blood cells. ---------------------------------------------------------------------------------------------------------- 10/17/2004 Extracted and Quantified RNA from gb_Larvae Label Vol Tri Extraction date Vol RNA Spec dil. A260 Conc. ug/ul E3 0.5 10/17/2004 10 1:99 0.194 0.776 E4a 0.5 10/17/2004 10 1:99 0.101 0.404 E4b 0.5 10/17/2004 10 1:99 0.252 1.008 E7 0.5 10/17/2004 20 1:299 0.897 10.764 E8 0.5 10/17/2004 15 1:199 0.532 4.256 E9 0.5 10/17/2004 15 1:99 0.347 1.388 E10 0.5 10/17/2004 20 1:99 0.5 2 E11 0.5 10/17/2004 20 1:99 0.406 1.624 E12 0.5 10/17/2004 20 1:99 0.368 1.472 E14 0.5 10/17/2004 15 1:99 0.162 0.648 E15 0.5 10/17/2004 10 1:99 0.161 0.644 10/18/2004 The following samples were analyzed with NVtmf/tmR/cap (dual label probe RT PCR) as follows: Samples: All Ozone treated haddock larvae(10/5 and 10/6) and gblarvae (10/17). Water 16.1 ul 2X Reaction Mix 25 ul 10 uM NVtmf 0.2 ul 10 uM NVtmr 0.2 ul 10 uM Nvcap (probe) 0.5 ul Dual Enzyme Mix 1 ul 0.2 ug/ul RNA 6 ul* Reaction volume 50 ul *3 ul of each positive control template was used ?one step 60?: 1. 50oC ? 30 minutes 2. 95oC ? 5 minutes 3. 95oC ? 15 seconds 4. 60oC ? 1 minute 5. Plate Read 6. GoTo Step 3, 39 more times 7. 21oC ? forever 8. End Results: No amplification in Negative Controls. Amplification in Positive Controls. No amplification in samples. ------------------------------------------------------------------------------------------------ 10/17/2004 Samples analyzed with Ps7L/Ps7R using SYBR Green RT-PCR system under the following conditions: Water 4.2 ul 2X Brilliant Green Master Mix 12.5 ul 10 uM Primer fwd 0.125 ul 10 uM Primer rev 0.125 ul RT RNase Block 0.0625 ul 0.2 ug/ul RNA 8 ul Reaction volume 25 ul Positive Control template: 1.5 ul NV+ eye gel ?Noda1?: 1. 50oC ? 30 minutes 2. 95oC ? 10 minutes 3. 95oC ? 30 seconds 4. 50oC ? 1 minutes 5. 72oC ? 30 minutes 6. Plate Read 7. GoTo Step 3, 39 more times 8. 95oC ? 1 minutes 9. 55oC ? 1 second; ramp 0.2oC/second 10. Melting Curve from 55-95oC, read every 0.5 oC, hold 30 seconds 11. Hold 21oC forever 12. End --------------------------------------------------------------------------------------