62209
Data Summary



Presentation

Jensen FISH495
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Final Report






5/18/2009
Real-time PCR with standardizing gene: 18s primers for O. mykiss.
Annealing temp = 55C


5/14/2009
Real-time PCR with primers for ACTH, CRH, NGF.
Annealing temp = 55C


5/7/2009
Ran a gel for the PCR reactions from 5/6.
external image 20090507.JPG
Gel Setup:
Ladder
 A1
 A2
 A3
 A4
 A5
 A6
 A7
 B1
 B2
 B3
 B4
 B5
 B6
 B7
 Ladder



Ladder
 C1
 C2
 C3
 C4
 C5
 C6
 C7











A = Primers for ACTH (Adrenocorticotropic hormone)
B = Primers for CRH (Corticotropin-releasing hormone)
C = Primers for NGF (Nerve Growth Factor)
1 = HE.B (not DNased)
2 = HE.B (DNased)
3 = HS.B (not DNased)
4 = HS.B (DNased)
5 = PS.B (not DNased)
6 = PS.B (DNased)
7 = Negative control (water)


5/6/2009
I ran (normal) PCR reactions for NGF, ACTH, and CH primers. The cDNA used were cDNA pools for each of my sampling groups, containing equal volumes from each individual sample. I also used cDNA pools made before I DNased the RNA samples to act as positive controls for the reactions (see gel on 4/9).


4/21/2009
Real time PCRs for all samples, using HSC71 primers with 3 water controls.
Note: Yesterday's qPCRs had abnormal melting curves, so used new Cyto13 that Sam made up. Hopefully that fixes the problem.


4/20/2009
I pooled some of my cDNA samples and then ran qPCRs with all 6 of my primer sets, for each pool. The pools contained equal volumes of four random samples within each given sampling category (HE, HS, PS).
The samples that were used to create each pool were:
HE: F1 F4 M2 M5
HS: F5 F6 M9 M12
PS: F24 F25 M22 M25

Real time PCR profile:
95C - 10 min
(95C - 30sec, 60C - 1min, 72C - 30sec) x 40
95C - 1 min


4/9/2009
I ran a gel on a 150ml 1% agarose gel for the 12 PCR products from 2/12/2009
Gel setup:
Ladder
 A-1
 A-2
 A-3
 A-4
 B-1
 B-2
 B-3
 B-4
 C-1
 C-2
 C-3
 C-4
 Ladder
A = Primers for ACTH (Adrenocorticotropic hormone)
B = Primers for CRH (Corticotropin-releasing hormone)
C = Primers for NGF (Nerve Growth Factor)
1 = Negative control
2 = HE.B (Hansen Creek, Entering)
3 = HS.B (Hansen Creek, Senescent)
4 = PS.B (Ponds, Senescent)

external image 20090409.JPG


4/7/2009
I ran qPCR's for all samples with primers for GH, HSC71, and GnRH, with 2 water controls for each primer set
The plate.


3/4/2009
I reverse transcribed all 30 DNased RNA samples.


2/25/2009
I DNased the 30 brain tissue RNA samples with the TURBO DNA-free kit.


2/24/2009
I finished the second part of the Chromogen Western blot
Membrane 1:
external image 20090224%20Blot_01.JPG
Membrane 2:
external image 20090224%20Blot_02.JPG


2/23/2009
I started a Western for 22 of my 28 liver samples. For the prepared samples that I chose to run on the gels (8 from HE samples, 7 from HS, & 7 from PS), I boiled them for 5 minutes and then centrifuged them at high speed for another 5 min. I then set up 2 gels, cleaned the wells and loaded them in this arrangement:
Gel 1:
Ladder
 M24
 M3
 M9
 M2
 F2
 F3
 M8
 M25
 M21
 F6
 F21
Gel 2:
F5
 F25
 M22
 F22
 Ladder
 F1
 M4
 F4
 M12
 M6
 M23
 F7
I then ran the gels for 45 minutes at 140 volts (machine would reset at higher voltages)

Gel 1:
external image 20090224%20Gel_01.JPG
Gel 2:
external image 20090224%20Gel_02.JPG

2/18/2009 - 2/19/2009
I quantified the 22 liver samples extracted on 2/17, using the Bradford method.
Average absorbance for each standard used to create a standard curve equation (y=0.0505*ln(x) - 0.2538):
Standard
 Absorbance
 Concentration
1
 0.128
 2000
2
 0.116
 1500
3
 0.095
 1000
4
 0.087
 750
5
 0.049
 500
6
 0.028
 250
Average absorbance for each sample and calculated concentration (ug/ml) from standard curve equation:
Tissue Sample
 Absorbance
 Concentration (μg/ml)
F3.L
 0.147
 2861.195
F5.L
 0.152
 3138.773
F7.L
 0.156
 3420.583
F22.L
 0.160
 3678.710
F25.L
 0.112
 1426.383
F2.L
 0.154
 3265.832
F21.L
 0.150
 3016.658
M2.L
 0.132
 2124.678
M3.L
 0.174
 4888.833
F24.L
 0.141
 2506.693
M25.L
 0.160
 3703.121
M22.L
 0.165
 4062.378
M8.L
 0.151
 3077.110
M6.L
 0.162
 3827.626
M4.L
 0.163
 3904.329
M23.L
 0.156
 3420.583
M9.L
 0.149
 2957.393
F4.L
 0.084
 814.342
F6.L
 0.132
 2096.759
M12.L
 0.159
 3630.370
M7.L
 0.155
 3331.277
M24.L
 0.152
 3118.083


2/17/2009
I extracted protein from the remaining 22 liver samples (2 liver samples could not be used because they were completely used for RNA extraction on 10/08/08: M5 & M10).


2/12/2009
I ran PCRs for the new primers that came in (ACTH, CRH, NGF), using the 3 brain tissue cDNA pools (HE.B, HS.B, PS.B)
PCR profile:
95C - 10 min
(95C - 45 sec; 55C - 45 sec; 72C - 30 sec) x40
72C - 10 min


2/4/2009 (continuation of Bradford method to quantify protein concentraions)
Standards measured to produce a standard curve (equation: y=0.0029x^0.5507):
Standard #
 ug/ml
 Absorbance
1
 2000
 0.174
1
 2000
 0.180
1
 2000
 0.210
2
 1500
 0.175
2
 1500
 0.161
2
 1500
 0.160
3
 1000
 0.135
3
 1000
 0.135
3
 1000
 0.132
4
 750
 0.111
4
 750
 0.113
4
 750
 0.112
5
 500
 0.087
5
 500
 0.085
5
 500
 0.084
5
 500
 0.082
6
 250
 0.060
6
 250
 0.062
6
 250
 0.065
Average absorbance for each sample and calculated concentration (ug/ml) from standard curve equation:
Tissue Sample
 Absorbance
 ug/ml
M1.L
 0.13
 997.684
M11.L
 0.134
 1054.126
M21.L
 0.127333
 960.832
F1.L
 0.157333
 1410.871
F8.L
 0.131
 1011.662
F23.L
 0.14
 1141.395


2/3/2009
I quantified the protein levels in each of the protein samples from 2/2, using the bradford method.


2/2/2009
I extracted protein from 6 liver samples (M1, M11, M21, F1, F8, F23), using 0.025g tissue and 500ul mammalian cell lytic solution.


1/29/2009
Real time PCR for cDNA from each pool (HE.B , HS.B , PS.B) for 3 different primer sets (GH , GnRH , HSC71). I did 3 water controls for each primer set.
PCR profile:
95C - 10 min
(95C - 30sec, 55C - 1min, 72C - 30sec) x 40
95C - 1 min


1/28/2009
I ran a gel for yesterday's PCR products:

Gel setup:
Ladder
 A-1
 A-2
 A-3
 A-4
 B-1
 B-2
 B-3
 B-4
 C-1
 C-2
 C-3
 C-4
 Ladder
A = Primers for GnRH (Gonadotropin-releasing hormone)
B = Primers for HSC71
C = Primers for GH (growth hormone)
1 = Negative control
2 = HE.B (Hansen Creek, Entering)
3 = HS.B (Hansen Creek, Senescent)
4 = PS.B (Ponds, Senescent)
external image 20090128.JPG
1/27/2009
I ran 3 PCRs for each cDNA pool (from 1/21) using primers for GnRH (gonadotropin-releasing hormone), HSC71 (heat-shock protein-like protein), and GH (growth hormone). The PCR profile was:
95C - 10 min
(95C - 45 sec; 55C - 30 sec; 72C - 30 sec) x40
72C - 10 min

I also made a 75ml 1% agarose gel to use tomorrow.


1/21/2009
I Reverse transcribed RNA pools from 1/20. 5ul from pool HE.B was used for the reaction since it had the lowest RNA concentration (for a total of 274ng RNA). Samples from the other 2 pools were diluted with water to equal the same total RNA.


1/20/2009
Made RNA pool for each group (1. Hansen Creek Entering, 2. Hansen Creek Senescent, 3. Ponds) with 464ng RNA from each sample.
Volume taken from each sample to reach 464ng was calculated by 464ng/RNA concentration of sample and is shown in the last column:

Code Location Status Sex ng/ul ul
M1 Hansen Creek Entering Male 25.54 18.17
M2 Hansen Creek Entering Male 285.57 1.62
M3 Hansen Creek Entering Male 18.56 25.00
M4 Hansen Creek Entering Male 201.57 2.30
M5 Hansen Creek Entering Male 266.88 1.74
M6 Hansen Creek Entering Male 41.69 11.13
M7 Hansen Creek Entering Male 79.24 5.86
F1 Hansen Creek Entering Female 211.81 2.19
F2 Hansen Creek Entering Female 39.35 11.79
F3 Hansen Creek Entering Female 165.85 2.80
M8 Hansen Creek Senescent Male 213.95 2.17
M9 Hansen Creek Senescent Male 252.57 1.84
M10 Hansen Creek Senescent Male 134.55 3.45
M11 Hansen Creek Senescent Male 154.46 3.00
M12 Hansen Creek Senescent Male 595.81 0.78
F4 Hansen Creek Senescent Female 62.69 7.40
F5 Hansen Creek Senescent Female 141 3.29
F6 Hansen Creek Senescent Female 171.66 2.70
F7 Hansen Creek Senescent Female 18.67 24.85
F8 Hansen Creek Senescent Female 38.62 12.01
M21 Iliamna Ponds Senescent Male 342.29 1.36
M22 Iliamna Ponds Senescent Male 328.8 1.41
M23 Iliamna Ponds Senescent Male 175.58 2.64
M24 Iliamna Ponds Senescent Male 91.07 5.09
M25 Iliamna Ponds Senescent Male 625.33 0.74
F21 Iliamna Ponds Senescent Female 372.43 1.25
F22 Iliamna Ponds Senescent Female 251.79 1.84
F23 Iliamna Ponds Senescent Female 291.38 1.59
F24 Iliamna Ponds Senescent Female 356.62 1.30
F25 Iliamna Ponds Senescent Female 380.28 1.22

I spec'd the completed pools to make sure they all had approximately the same total RNA:


1/15/2009
Finished RNA isolation from 1/12. F4 M6 & M24 had chloroform carryover and small, visible pellets. M7 showed a large pellet and no chloroform carryover after the precipitation step. All had clearly visible pellets after RNA wash.

Also ran specs for RNA from today, 11/24, & 11/21 so that all 30 brain tissues samples have now been spec'd. Note: M21.B2 is the correct spec for M21 (not M21.B).


1/12/2009
Isolated RNA from the final 4 O. nerka brain tissue samples.
Starting tissue weights:
(F4) HS.F4.B = .41g
(M6) HE.M6.B = .31g
(M7) HE.M7.B = .43g
(M24) PS.M24.B = .39g
I homogenized the tissues in .8ml TRI-Teagent and then removed half of the homogenate to archive with other samples at -20C. 2.1ml additional TRI-Reagent was added to each tube not archived. After this step, I followed standard TRI-Reagent protocol x2.5 (used .5ml Chloroform for RNA extraction step and 1.25ml Isopropanol for the RNA precipitation step). Had to leave after RNA precipitation step, so I stored the samples at -20C (on top shelf) after they were spun.


11/24/2008
Isolated RNA from 8 O. nerka brain tissue samples.
Starting tissue weights:
(F21) PS.F21.B = .27g
(F22) PS.F22.B = .34g
(F23) PS.F23.B = .32g
(F24) PS.F24.B = .49g
(F25) PS.F25.B = .59g
(M21) PS.M21.B = .30g
(M22) PS.M22.B = .50g
(M23) PS.M23.B = .42g
I homogenized the tissues in .8ml TRI-Reagent and then removed half of the homogenate to archive with other samples at -20C. 2.6ml additional TRI-Reagent added to each tube. After this step, I followed standard TRI-Reagent protocol x3. All samples, except for M21, showed chloroform carryover after RNA precipitation step, and pellets were small or not visible. I continued with protocol, removing chloroform with rest of supe before adding 75% EtOH. After spin, pellets were still light in most tubes but visible in all tubes.


11/21/2008
Isolated RNA from 8 more O. nerka brain tissue samples.
Starting tissue weights:
(F1) HE.F1.B = .44g
(F2) HE.F2.B = .43g
(F3) HE.F3.B = .45g
(M1) HE.M1.B = .58g
(M2) HE.M2.B = .56g
(M3) HE.M3.B = .52g
(M4) HE.M4.B = .49g
(M5) HE.M5.B = .48g
I homogenized the tissues in .8ml TRI-Reagent and then removed half of the homogenate to archive with other samples at -20C. Added 2.6ml of additional TRI-Reagent to bring the volume of TRI-Reagent in each tube up to 3ml. Added .6ml chloroform to each tube and incubated at RT for 15 minutes. Spun at 4C. Precipitated RNA with 1.5ml isoprpanol. spun. All samples had chloroform carryover. Chloroform was removed and samples re-spun. Only a few, small pellets were visible and still some chloroform present. Poured off supe and added 1ml 75% ethanol and spun 7500xg for 5min. Sam took over after this step.


11/19/2008
Began isolating RNA from O. nerka brain tissues using TRI-Reagent method
Starting tissue weights:
(F5) HS.F5.B = .38g
(M11) HS.M11.B = .37g
(F8) HS.F8.B = .52g
(M10) HS.M10.B = .39g
(M12) HS.M12.B = .34g
(F6) HS.F6.B = .36g
(F7) HS.F7.B = .27g
(M8) HS.M8.B = .34g
*Only used half of tissue volume after homogenizing the tissue with 1ml TRI-Reagent. Archived other half with rest of samples at -20C.
After removing half of volume, I followed TRI-Reagent protocol X2.5

- kubu4 kubu4 Sam took over after isopropanol precipitation spin. Virtually no detectable pellets. Some chloroform carryover from the previous step was present and was removed. Added 1.5mL of additional isopropanol to each tube, mixed and spun 12,000xg for 8 mins. 4C. Repeated isopropanol precip because volume of samples appeared to be small (~3mL). After spin, more pellets were visible, but also more chloroform. Chloroform was removed and the tubes spun again. Supe was removed, pellets washed with 1mL 75% EtOH and spun 7500xg, for 5mins. 4C. Supe was removed and pellets air dried for 10mins. 50uL water was used to resuspend RNA. Samples were transferred to 1.5mL snap cap tubes and incubated 55C for 10mins. Samples were spec'd and then stored @ -80C in a yellow rack (TEMPORARILY) on the second shelf down from the top.

external image 20081119%20RNA%20LJ.bmp
NOTE: The second "M12" reading is the correct one. The first reading I left the arm up.


11/10/08
I ran a gel with the PCR products from 11/07
Gel Setup:
Ladder
 HS.M10.L (1)
 HE.M2.M (1)
 PS.M25.B (1)
 PS.F23.M (1)
 HE.M5.L (1)
 HS.M9.B (1)
 CNTRL (1)
 HS.M10.L (2)
 HE.M2.M (2)
 PS.M25.B (2)
 PS.F23.M (2)












Ladder
 HE.M5.L (2)
 HS.M9.B (2)
 CNTRL
(2)
 HS.M10.L (3)
 HE.M2.M (3)
 PS.M25.B (3)
 PS.F23.M (3)
 HE.M5.L (3)
 HS.M9.B (3)
 CNTRL (3)
(1) = GnRH primers (expected product size = 226 bp)
(2) = GH primers (expected product size = 312 bp)
(3) = HSC71 primers (expected product size = 217 bp)

external image 20081110.png


11/07/2008
Using the 6 cDNA samples from 11/03, I ran 3 PCRs for each sample using primers for GnRH (gonadotropin-releasing hormone), HSC71 (heat-shock protein-like protein), and GH (growth hormone). The PCR profile was:
95C - 10 min
(95C - 30 sec; 50C - 30 sec; 72C - 2 min) x40
72C - 10 min

Gel setup:
Ladder
 HS.M10.L (A)
 HE.M2.M (A)
 PS.M25.B (A)
 PS.F23.M (A)
 HE.M5.L (A)
 HS.M9.B (A)
 CNTRL (A)
 HS.M10.L (B)
 HE.M2.M (B)
 PS.M25.B (B)
 PS.F23.M (B)
 HE.M5.L (B)
 HS.M9.B (B)
 CNTRL (B)
(A) = O. mykiss 18s primers
(B) = MSTN lb qPCR primers
CNTRL = No template

external image 20081107.png

11/03/2008
I reverse transcribed the O. nerka RNA samples isolated on 10/08 and ran 2 PCRs for each sample using primers from Sam for O. mykiss 18s (PCR-A) and MSTN lb qPCR (PCR-B). The PCR profile was:
94C - 2 min.
(94C - 1 min; 50C - 2 min; 72C - 2 min) x40
72C - 10 min

Sample Codes:
HE = Hansen Creek, sample collected as fish was ENTERING the stream
HS = Hansen Creek, sample collected after fish showed strong signs of SENESCENCE (ie frayed fins, loss of coloration, weakness, loss of aggression, etc.)
PS = Iliamna Ponds, sample collected after fish showed signs of SENESCENCE
F# = Female + number assigned to each individual fish
M# = Male + number assigned to each individual fish
L = Liver tissue
M = Muscle tissue
B = Brain tissue
Example: HS.M10.L = Hansen Creek, senescent, male 10, liver tissue

10/08/2008
I isolated RNA using the TRI REAGENT method for 6 samples of Oncorhynchus nerka tissue.

Notes (added 10/15/08): brain tissue samples did not show pellets during RNA isolation steps. Liver samples had very low numbers for 260/230, so I added 200ul more water to and tested again. This improved the number, but still low so I added 200ul water again, producing better results (though still low for sample HE.M5.L (HE.M5.L2 = sample after first addition of extra water; HE.M5.L3 = after second addition of extra water)

Starting weights:
HS.M10.L = .56g
HE.M2.M = .60g
HE.M5.L = .94g
PS.F23.M = .55G
HS.M9.B = .19g
PS.M25.B = .38g
external image 20081008%20%20RNA%20LJ.bmp

9/26/2008
I isolated RNA using the TRI REAGENT method from 50mg of tissue.