5 5 2015 310 Heat Shock RNA Isolation / qPCR

Today I process the heat shock samples from the Fish 310 lab. I isolated the RNA, DNase treated the sample, and ran a qPCR for quality control. I changed a few things from the previous protocols to make things run smoother and more efficient today. I've also taken several of Sam's suggestions on how I can improve my notebook.

RNA Isolation Protocol.

1. 100 mg tissue homogenized in 1 ml RNAzol RT at room temp. 
2. Added 400 ul 0.1% DEPC water to homogenate.
3. Incubated 15 minutes at room temp
4. Centrifuged 15 minutes at 11,400 rpm (~12,000 g)
5. Transferred 1 ml supernatant to fresh tube
6. Added 400 ul 75% EtOH
7. Mixed via inversion for 15 seconds
8. Incubated samples 10 minutes at room temp
9. Centrifuged 8 minutes at 11,400 rpm (~12,000 g)
10. Discarded supernant
11. Added 600 ul 75% EtOH
12. Vortexed 15 seconds
13. Centrifuged 3 minutes at 7,400 rpm (~8000 g)
14. Repeated steps 10 - 13. 
15. Removed supernatant
16. resuspended RNA in 50 ul 0.1% DEPC water

Instead of nanodropping the RNA, I immediately Turbo DNase Treated the samples to remove any DNA contamination. 

DNase Treatment Protocol:

1. Added 5 ul DNase 10X Buffer to each 50 ul of RNA.
2. Added 1 ul Turbo DNase to each RNA.
3. Incubated at room temp for 30 minutes
4. Added 5 ul DNase inhibitor to each sample
5. Incubated at room temp for 5 minutes, flicking occasionally
6. Centrifuged at 9400 rpm (~10,000 g) for 1.5 minutes
7. Decanted the top 50 ul of supernatant to fresh tube

8. Nanodropped

Nanodrop Results (including positive control):

Sample ID	Date	Time	ng/ul	A260	A280	260/280	260/230
C+	5/5/2015	4:17 PM	167.3	4.182	2.092	2	2.34
42715HT1	5/5/2015	4:18 PM	238.58	5.965	3.366	1.77	0.66
42715HT2	5/5/2015	4:19 PM	422.45	10.561	5.431	1.94	1
42715HT3	5/5/2015	4:21 PM	175.57	4.389	2.26	1.94	1.79
42715HT4	5/5/2015	4:22 PM	251.19	6.28	3.532	1.78	0.68
42715HT5	5/5/2015	4:23 PM	391.29	9.782	5.133	1.91	0.87
42715ST1	5/5/2015	4:24 PM	690.94	17.274	10.258	1.68	0.49
42715ST2	5/5/2015	4:25 PM	393.97	9.849	5.181	1.9	0.91
42715ST3	5/5/2015	4:25 PM	365.73	9.143	4.917	1.86	0.99
42715ST4	5/5/2015	4:26 PM	426.33	10.658	5.407	1.97	0.99
42715ST5	5/5/2015	4:27 PM	343.49	8.587	4.622	1.86	0.81

For the qPCR I used Actin primers and a positive control from Fidalgo seed oysters extracted on 3/23/2015. My negative control contained no DNA as to show there was no contamination in the Master Mix. I also bumped up the volume of sample used to 0.5 ul due to issues with the pipetter not being able to accurately pipette 0.2 ul. This is about 2.5 X the concentration of contamination gDNA that the cDNA will have in it. I also bumped down the amount of water by 1 ul per reaction so as to ensure proper reaction mixture ratios were maintained. 

qPCR Reagent Table:

	Volume	Reactions X12
Ssofast Evagreen MM	10	140
FWD Primer	0.5	7
REV Primer	0.5	7
Nuclease Free H2O	8.8	123.2
RNA	0.5

qPCR protocol:

1. Added each from greatest volume to least to make the master mix. 

2. Pipetted 20 ul master mix into each well of a qPCR partial plate

3. Added 0.5 ul sample to each tube

qPCR plate lay out:

	11	12
C	C-	C+
D	42715HT1	42715ST1
E	42715HT2	42715ST2
F	42715HT3	42715ST3
G	42715HT4	42715ST4
H	42715HT5	42715ST5

This layout can be seen in the top left corner of the qPCR image below. 

qPCR program:

Sybr New Plate+Sybr cDNA 55 melt 1 Read
Step	Temperature	Time
Initiation	95 C	10 min
Elongation	95 C	15 sec
	55 C	15 sec
Read
	72 C	15 sec
Repeat Elongation 40 times
Termination	95 C	1 min
	55 C	1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C	65 - 95 C	30 sec
	21 C	10 min
End

Results:

It looks like the DNase treatment removed some but not all the DNA. The DNA replicates much much earlier in the cycles. Overall I think these samples are good samples with high concentrations and mild DNA contamination. You can see the data files for the qPCR here and here. 

Sometime this week I will begin the process of creating cDNA to use with qPCR to test for HSP70 and other RNA markers. 

5 4 2015 DNase treatment

Today I ran the DNase treatment on the samples to remove any excess DNA from the samples. The treatment was pretty simple but I don't think it helped much as their was amplification at roughly the sample cycle as there was in the last quality check. The DNase treatments were old but I don't think that would cause an issue. I also had to mix kits as there wasn't enough reagents in a single kit to do the treatments on 10 samples.

Protocol for treatment:

1. Added 10 ul DNase 10X Buffer to each 100 ul of RNA.
2. Added 2 ul Turbo DNase to each RNA.
3. Incubated at 37 C for 30 minutes
4. Added 10 ul DNase inhibitor to each sample
5. Incubated at room temp for 5 minutes, flicking occasionally
6. Centrifuged at 10,000 g for 1.5 minutes
7. Decanted the top 100 ul of supernatant to fresh tube
8. Placed in -80 until I could run qPCR

Later this afternoon I ran the qPCR on the treatment.

Reagent work sheet:

	Volume	Reactions X12
Ssofast Evagreen MM	10	140
FWD Actin Primer	0.5	7
REV Actin Primer	0.5	7
Nuclease Free H2O	9.8	137.2
RNA	0.2

1. Added each from greatest volume to least to make the master mix. 

2. Pipetted 21 ul master mix into each well of a qPCR partial plate

3. Added 0.2 ul sample to each tube

4. Positive control was DNA extract from Oly seed oysters

5. Negative control had nothing added to the master mix

I ran the following program:

Sybr New Plate+Sybr cDNA 55 melt 2
Step	Temperature	Time
Initiation	95 C	10 min
Elongation	95 C	15 sec
	55 C	15 sec
Read
	72 C	15 sec
Read
Repeat Elongation 40 times
Termination	95 C	1 min
	55 C	1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C	65 - 95 C	30 sec
	21 C	10 min
End

You can see the results below

bright green = positive control, red = negative control, light blue down to brown = HM1-5, dark blue to yellow = SM1-5

The positive control began amplifying at the 21 cycle marks. One sample began at 30 cycles the rest were between 34 and 36. 

You can see the raw datafile here.

Tomorrow I will isolate the RNA from the 310 heat shock samples, treat with DNase, and qPCR for quality. 

May 2015 Goals

In an effort to keep myself on track to compete the SAFS masters program by later this year I am going to write monthly goals to complete for each month.

This month:

Process samples to produce cDNA for qPCR
Run qPCR using primers for HSP70, Actin, and other interesting primers
Edit the Materials and Methods section
Write the Results sections of my next manuscript
Begin writing the Intro/Discussion
Prepare for my wedding

5 1 2015 qPCR Quality Check

Today I ran a quality check on the samples that I isolated yesterday. Instead of creating cDNA before doing the qPCR I ran just the RNA with Actin primers to determine if there was any genomic contamination in the RNA samples. I diluted the RNA to levels similar to that which would have gone from the cDNA process to the qPCR process. This was to ensure that any genomic dna would be at levels similar to that from from the cDNA process and wouldn't over estimate the concentration of them in the sample.

The qPCR protocol was as follows

Master Mix

	Volume	Reactions X12
Ssofast Evagreen MM	10	120
FWD Primer	0.5	6
REV Primer	0.5	6
Nuclease Free H2O	9.8	117.6

1. Added the reagents from highest volume to lowest to the master mix

2. Vortexed

3. pipetted into .5 ml qPCR tubes

4. Added 0.2 ul RNA to each tube from each isolation

5. Placed into the Opticon 2 qPCR machine

6. Ran the program "Sybr New Plate + Sybr cDNA 55 melt 2 reads"

Results

As you can see, the samples began amplifying at 27-30 cycles which is indicative of a genomic DNA contamination. The negative control did not begin amplifying until 37 cycles. 

For future work, I need to use a DNase treatment to remove genomic DNA before I create cDNA. Next week I will isolate the 310 heat shock animals, DNase treat everything, and produce cDNA. 

4 30 2015 RNA Isolation FISH 310 samples

Today I isolated RNA from samples collected by the Biology of Shellfish students. These samples are nearly identical to mine except that they were treated slightly different and collected by the students. The tissue is primarily ctenidia but there were traces of mantle and glycogen stores in it. The samples were collected on tuesday and stored in RNAzol in the -80 until today.

The samples were labeled
42815HM1-5
42815SM1-5

The protocol is as follows:

1. 100 mg tissue homogenized in 1 ml RNAzol RT at room temp. 
2. Added 400 ul Nanopure water to homogenate.
3. Incubated 15 minutes at room temp
4. Centrifuged 15 minutes at 11,400 rpm (~12,000 g)
5. Transfered 1 ml supernatant to fresh tube
6. Added 400 ul 75% EtOH
7. Mixed via inversion for 15 seconds
8. Incubated samples 10 minutes at room temp
9. Centrifuged 8 minutes at 11,400 rpm (~12,000 g)
10. Discarded supernant
11. Added 600 ul 75% EtOH
12. Vortexed 15 seconds
13. Centrifuged 3 minutes at 7,400 rpm (~8000 g)
14. Repeated steps 10 - 13. 
15. Removed supernatant
16. resuspended RNA in 100 ul Nanopure water
17. Nanodropped

The results from the nanodrop were as follows. 

Sample ID	Date	ng/ul	A260	A280	260/280	260/230
42815HM1	4/30/2015	94.49	2.362	1.336	1.77	2.25
42815HM2	4/30/2015	46.68	1.167	0.741	1.57	0.88
42815HM3	4/30/2015	40.23	1.006	0.649	1.55	0.67
42815HM4	4/30/2015	105.78	2.644	1.475	1.79	1.43
42815HM5	4/30/2015	182.93	4.573	2.517	1.82	2.19
42815SM1	4/30/2015	257.87	6.447	3.468	1.86	2.29
42815SM2	4/30/2015	66.35	1.659	0.999	1.66	2.26
42815SM3	4/30/2015	43.2	1.08	0.645	1.67	0.99
42815SM4	4/30/2015	143	3.575	2.001	1.79	1.24
42815SM5	4/30/2015	235.27	5.882	3.195	1.84	1.69

Tomorrow I will run a qPCR on the RNA to verify the purity of the RNA and ensure there is no genomic DNA in the sample. 

4 23 2015 Heat/Mechanical Shock 24 Hour Time Point

Today I completed the 24 hour time point collection and the collection of the controls for the experiment. Tonight I ran out of liquid nitrogen before doing the control groups. I put them on ice and placed them in the -80 ASAP.

Heat Stress

• 24 hours post exposure 8 animals each pop staggered by 1 hour
  • N pop first, H pop next, S pop last
  • collected with ethanol flame sterilized forceps and scissors
    • tools kept in 100% EtOH
    • ran through EtOH flame
    • allow EtOH to burn off
    • Collect tissue
    • replace in 100% EtOH
    • wipe occasionally with paper towel and resterilize
  • ctenidia sample (50-100 mg) homogenized in 1 ml RNAzol stored at -80°C
  • 2nd ctenidia sample in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • mantle sample in 1.5 ml screw cap tube dropped into liquid nitrogen -80°C
  • remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
  • shells placed in labeled plastic bags so correspond to sample including oyster id

Mechanical Stress

• 24 hours post exposure 8 animals each pop staggered by 1 hour
  • N pop first, H pop next, S pop last
  • collected with ethanol flame sterilized forceps and scissors
    • tools kept in 100% EtOH
    • ran through EtOH flame
    • allow EtOH to burn off
    • Collect tissue
    • replace in 100% EtOH
    • wipe occasionally with paper towel and resterilize
  • ctenidia sample (50-100 mg) homogenized in 1 ml RNAzol stored at -80°C
  • 2nd ctenidia sample in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • mantle sample in 1.5 ml tube dropped into liquid nitrogen - then to -80°C
  • remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
  • shells placed in labeled plastic bags so correspond to sample including oyster id

Controls

For controls sample 8 from each group, this should be done on day 2 as less things are going on. Protocol is the same. These oyster should have remained in holding tank-throughout and should simply be removed and sampled. There is no treatment. Using sterile techniqes.

• ctenidia sample (50-100 mg) homogenized in 1 ml RNAzol stored at -80°C
• 2nd ctenidia sample (any remaining) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
• mantle sample (50-100 mg) in 1.5 ml tube dropped into liquid nitrogen then to -80°C
• remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
• shells placed in labeled plastic bags so correspond to sample including oyster id

4 22 2015 Heat/Mechanical Shock Experiment

Today I ran the Heat and Mechanical Shock experiment on my oysters. While most groups got liquid nitrogen treatment, the mechanical H and S groups did not due to lack of Liquid N2. For those samples I placed them on ice and moved them to the -80 as soon as possible.  The procedure went as follows.

Pre-Experiment

Oysters

• 8°C
• fed daily with 5-10 ml per tank commercial shellfish diet (Isochrysis 1800 mix Marine Algae)
• Partial water changes once per week (remove 5 gallons, replace with fresh 5 gallons)
  • Seawater from Seattle Aquarium
• Aeration with aquarium aerator/stone
• circulation with underwater pump
• Tanks 10 gallon plastic storage tubs (costco storage tubs)

Homogenization tubes for ctenidia prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, and oyster # (1,2,3...)

Collection 1.5 ml tubes (screw cap) for ctenidia prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, oyster # (1,2,3...), and tissue type (C, M)

Collection 1.5 ml tubes (screw cap) for mantle prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, oyster # (1,2,3...), and tissue type (C, M)

15 ml conical tubes for whole oyster prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, and oyster # (1,2,3...)

Warm fresh seawater in 500 ml beaker to 38°C by immersing it in a water bath at 38°C.

Experiment

Heat Stress

• 22 oysters each pop in a mesh bag were lowered (stagger each pop by 1 hour) into 500 ml of prewarmed 38°C sea water (1 hr timer started)
  
  • N pop first, H pop next, S pop last
• after 1 hour exposure heat stress ends, animals returned to 8°C water.
• 1 hour post exposure 8 animals sampled from each group staggered by 1 hour
  • N pop first, H pop next, S pop last
  • collected with ethanol flame sterilized forceps and scissors
    • tools kept in 100% EtOH
    • ran through EtOH flame
    • allow EtOH to burn off
    • Collect tissue
    • replace in 100% EtOH
    • wipe occasionally with paper towel and resterilize
  • ctenidia sample (50-100 mg) homogenized in 1 ml Trizol stored at -80°C
  • 2nd ctenidia sample (any remaining) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • mantle sample (decent size) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
  • shells in individual labeled ziploc bags so correspond to sample including oyster id

Mechanical Stress

• 22 oysters from 1 pop placed in bucket in centrifuge and spun for 5 minutes at 1000 rpm.  Samples staggered by 1 hour. Completed after all heat shock treatments and samples were taken.
  • N pop first, H pop next, S pop last
• after 5 minutes of mechanical stress, animals returned to 8°C water
• 1 hour post exposure 8 animals sampled from each group staggered by 1 hour
  • N pop first, H pop next, S pop last
  • collected with ethanol flame sterilized forceps and scissors
    • tools kept in 100% EtOH
    • ran through EtOH flame
    • allow EtOH to burn off
    • Collect tissue
    • replace in 100% EtOH
    • wipe occasionally with paper towel and resterilize
  • ctenidia sample (50-100 mg) homogenized in 1 ml Trizol stored at -80°C
  • 2nd ctenidia sample (any remaining) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • mantle sample (decent size) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
  • shells in individual labeled ziploc bags so correspond to sample including oyster id