Protocol test: Round 4

Today, Kaitlyn fulfilled her dream of getting to do labwork on the weekend (hey, she volunteered to help even after I told her she didn’t need to). As I mentioned in my planning post, we followed the PAXgene protocol to extract DNA from histology blocks with three changes:

• We had 2 methods for lysis: TissueTearor at setting 1 for 10 seconds, or vortexing with glass beads for two minutes. This is slightly different from the previous TissueTearor methods. I thought I should use a milder TissueTearor setting so I can compare DNA shearing across methods with the Seeb’s BioAnalyzer.
• We no longer have the Thermomixer. Instead, we’re incubating the tubes on a heat block for 10 minutes, then vortexing at maximum speed for one minute. Total incubation time is still 60 minutes.
• I’m no longer removing RNA from the DNA I’m extracting. I would like to isolate DNA and RNA from the same samples so I can compare epigenetic and transcriptomic information.

Figure 1. Heat block incubation set-up.

I am using four tissue samples — split between two tubes for each method — with eight samples total. Kailyn pre-scraped two samples on Friday (see her lab notebook here) and scraped the other two samples this morning (IDs here). Because the tissue is already preserved in wax, I wanted to see if scraping tissue into a tube beforehand affected DNA yield. Tissue scraping is a rate-limiting step, so being able to scrape out tissue the night beforehand would be a real timesaver!

Method notes

• Step 9: We incubated the tubes at 37ºC for 25 minutes so the ethanol was fully evaporated
• Step 12-13: I used the TissueTearor at setting 1 for 10 seconds in the sample. Before use and after samples, I cleaned the TissueTearor in ethanol for 20 seconds, then rinsed in two different batches of DI water for 20 seconds each.
• Step 15: We incubated the tubes on the heat block at 56ºC for ten minutes, then vortexed for one minute. We repeated this process 6 times for a total incubation time of one hour. There was no gelatinous pellet, so the proteinkinase K digestion must have gone well!
• Step 17: Skipped!
• Step 18: The tubes were kept at room temperature for 3 minutes while the heat block reached temperature. We incubated tubes on the heat block at 80ºC for ten minutes, then vortexted for one minute. We repeated this process 6 times for a total incubation time of one hour.
• Step 27: No DNA came out of the T4-V2 and T6-TT tubes! I think there might have been something clogging the spin column…?
• Step 28: I made a working solution wiht 2626.8 µL of buffer and 13.2 µL of dye. S1 = 161.34; S2 = 9957.65

Results

Table 1. Initial mass and DNA concentrations for tissue samples processed.

Tube	Initial Mass (g)	DNA Concentration (ng/µL)
T4-TT	0.0207	3.06
T4-V2	0.0207	N/A
T5-TT	0.0204	7.66
T5-V2	0.0207	N/A
T6-TT	0.0210	N/A
T6-V2	0.0214	2.04
T7-TT	0.0214	N/A
T7-V2	0.0210	N/A

I got DNA, even without a Thermomixer! My yields, however, were pretty inconsistent. They were either 2 ng/µL and above, or N/A. I think part of the problem was the initial mass of tissue: Kaitlyn went above 0.0200 g, maybe because I wasn’t specific about being as close to 0.0200g as possible. The TissueTearor method worked better than the vortexing method. I think the big takeaway here is still variability between tissues.

Going forward

Well, I know the method still works without the Thermomixer! If I’m doing a different bisulfite assay that only needs 1 ng/µL of DNA, I can definitely move forward with my samples. If not, it’s back to testing.