Tuesday, May 5, 2015

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 IDDateTimeng/ulA260A280260/280260/230
C+5/5/20154:17 PM167.34.1822.09222.34
42715HT15/5/20154:18 PM238.585.9653.3661.770.66
42715HT25/5/20154:19 PM422.4510.5615.4311.941
42715HT35/5/20154:21 PM175.574.3892.261.941.79
42715HT45/5/20154:22 PM251.196.283.5321.780.68
42715HT55/5/20154:23 PM391.299.7825.1331.910.87
42715ST15/5/20154:24 PM690.9417.27410.2581.680.49
42715ST25/5/20154:25 PM393.979.8495.1811.90.91
42715ST35/5/20154:25 PM365.739.1434.9171.860.99
42715ST45/5/20154:26 PM426.3310.6585.4071.970.99
42715ST55/5/20154:27 PM343.498.5874.6221.860.81
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:
VolumeReactions X12
Ssofast Evagreen MM 10140
FWD Primer0.57
REV Primer0.57
Nuclease Free H2O8.8123.2

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:

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
Initiation95 C10 min
Elongation95 C15 sec
55 C15 sec
72 C15 sec
Repeat Elongation 40 times
Termination95 C1 min
55 C1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C65 - 95 C30 sec
21 C10 min

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. 


  1. Big picture questions regarding your results:

    If you still have gDNA contamination in your RNA, why would you proceed to making cDNA? If you make cDNA from these samples, how will you know if amplification produced in qPCRs run using the cDNA is due to residual gDNA or from the cDNA? Additionally, if you're OK with having contaminating gDNA in your RNA, why perform the DNasing in the first place?

    Other things I noticed:

    - Your qPCR reactions still don't add up to 20uL. Additionally, there's some discrepancy in your notebook as to what the final volume of the qPCR reactions actually is. The table shows a final volume of 20.3uL but in the text you say that you added 0.5uL to 20uL of master mix - final volume 20.5uL. As I mentioned previously, the degree of deviation from the proper final volume probably has no noticeable impact on your reactions, but you should correct it in future qPCRs.

    - You're still using the term "negative control", but I think you mean "no template control." Your notebook also isn't clear on whether you substituted water for DNA template (which is necessary to have a proper no template control). An example of a negative control would be to use C. gigas DNA in a qPCR reaction instead of O. lurida DNA. A no template control uses water as template instead of any DNA.

  2. Oh, one more thing. I noticed that on your qPCR run from yesterday (http://goo.gl/kqshoz) and this one that your melt curves did not complete. Did you happen to stop the program before it finished on both runs? The melt curve protocol should go to 95C, but on yesterday's it stops at ~69C and on this one it stops at ~71C.

  3. Sorry, just one more thing I notice; I promise!

    Your centrifugation numbers are wrong. The g-force values you have listed don't match the RPMs (or, vice versa, but I think you were using the RPM setting on the centrifuge yesterday).

    On the centrifuge you were using 7400RPM = 5143g. 11400RPM = 12205g.

    I'd recommend just using the RCF (g) setting on the centrifuge.

    There's really no reason to use (nor report) RPMs, since RPM values will have different corresponding RCFs across different centrifuges (RCFs are dependent upon the radius of the rotor in the centrifuge).