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.