DNasing RNA, Round II-V

DNasing, Rounds II-V

I performed 3 rounds of DNase reactions (n=30 per round) on the Oly larval and ctenidia RNA. I followed the same protocol as Round 1, using 50 uL RNA for each sample, 5 uL buffer, 1 uL DNase, and 5 uL inactivation reagent.

There were two samples - 38 and 46 (extracted previously in 2018) - which had less than 50 uL total volume in the original RNA sample. For these I used 25 uL RNA, 2.5 uL buffer, 1 uL DNase and 2.5 uL inactivation reagent.

Several of the samples also required dilution prior to DNasing, since the maximum amount of RNA in a 50 uL reaction is 10 ug. For these I added 50 uL DEPC-treated water and vortexed to mix, prior to DNasing: 477, 43, 564, 291, 304, 306, 309, 315, 316, 317, 321, 325, 335, 345.

Some images of the Turbo DNase process

This is the Turbo DNase kit I used.

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In it is: A) Buffer B) TurboDNase enzyme C) Inactivation Reagent D) Ultrapure water (to resuspend inactivation reagent, if it has dried).

NOTE: the kit says it’s good for 50 reactions, but that’s for 100 uL volume / reaction. For me, the limiting factor was the DNase enzyme - there was 120 uL DNsae (used 1 uL per sample) - so I did ~110 samples with one kit before pulling DNase from the other.

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2 sets of labeled 0.5 mL tubes. Step 1 is to transfer 50 uL RNA into one set of tubes.

After adding buffer (10% of RNA volume, in my case 5 uL), and DNase (1 uL), I vortexted gently and incubated at 37C for ~25 minutes in the Thermo Cycler. It takes no time to heat up, keeps track of the time. Maximum # tubes at once is 30.

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I forgot to get images of the inactivation reagent step - the solution is milky white, and after mixing and incubating at rooom temp for 5 minutes, samples are centrifuged to “pellet” the reagent, then the supernatant containing RNA is transferred to the 2nd batch of tubes. NOTE: I held everything on ice while working.

qPCR to check for DNA contamination

I ran 3 more batches of qPCR to check for DNA contamination.

For these batches, I selected Olympia oyster primers ordered by Jake

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Primers are located in the freezer in FTR 213.

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Found the Oly primers using the database.

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I created working stocks of my primers - diluted 12 uL each primer in 108 uL DECP-treated water, creating 120 uL at 10 uM for each.

Created a mastermix for all reactions using the following calculations, then pipetted 19 uL mastermix onto qPCR plates, and 1 uL each sample in duplicate.

Date 1 plate , 32 rxns All 3 reactions
# Samples 32 96.0
# Reactions 68 204.0
Template (RNA sample) (uL) 68.0 204.0
Sso Fast (uL) 748.0 2,244.0
Pf, 10 uM (uL) 37.4 112.2
Pr, 10 uM (uL) 37.4 112.2
DEPC-treated water (uL) 598.4 1,795.2
Total Volume in mastermix (uL) 1,421.2 4,263.6

Round II

Results look good - only the positive control (DNA sample 1A, in green) had a detected melt temperature. I realized later that I accidentally used the wrong qPCR protocol - I used “CFX_2StepAmp60_EVAGreen+Melt.prcl” instead of “CFX_2StepAmp_EVAGreen+Melt.prcl”. The difference is that step 3 is 60C, while in the correct protocol step 3 is 55C. For my purposes, and since results look good, it seems that this error does not warrant a re-do. I’ll check with the lab, though.

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Round III

Results look a bit weird, different than before. The fluorescence (RFU) goes up at high temperatures - not sure if this indicates an issue. Also, two samples had melt temperatures - 543 and 475 - but only in 1 rep for each.

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Round IV

Results look okay, except for one well again had a weird increase in RFU at high temps (sample 476, rep 2). However, there was no melt temperature measured in the RNA samples.

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All edited CFX files (with sample names, colors) are located in the qPCR-DNA-contamination directory in my O.lurida_Stress repo.

Raw CFX files are saved to owl here

Written on August 5, 2019