According to the QuantSeq library prep protocol, I need to ensure no DNA contamination in my RNA samples. Sam advises that all RNAzol-processed samples will definitely have some DNA contamination. So, I am using the Turbo DNase kit to clean my RNA.

July 31st - DNased RNA, qPCR to identify DNA contamination, batch 1 (n=30)

I ran a first batch of reactions (n=30) using the Turbo DNase kit. The kits we had in the -20 freezer were old (from 2014), so I walked over to the BioSciences stock room (J wing) and purchased 2 new kits (50 reactions per; I’ll need a couple more kits). NOTE: each kit is ~$145.

Turbo DNA Protocol

All reactions used 50 uL RNA, as per the manual’s example. Here were my steps:

• Labeled 2 sets of 0.5 mL microcentrifuge tubes with RNA sample names
• Transfered 1 uL of DNase into one set of tubes
• Transfered 5 uL of Turbo DNase Buffer into each tube
• Transfered 50 uL of RNA into tubes
• Mixed gently - used low setting on the vortexer.
• Incubated at 37C - used the thermocycler in FTR 209 - for 20 minutes
• Removed samples from thermocycler, added 5 uL inactivation reagent solution. Prior to pipetting this inactivation reagent, I vortexted it thoroughly.
• Incubated at room temperature for 5 minutes. Vortexed each sample briefly twice during this incubation time to keep solution mixed.
• Centrifuged samples for 90 seconds at 10,000 rcf
• Carefully transferred supernatant to fresh, labeled tubes.
• Held DNased RNA on ice, while I learned how to run qPCR

qPCR to assess DNA contamination

I will use the SsoFast enzyme, a DNA polymerase technology that performs a super fast reaction, and the BIORAD CFX Connect qPCR machine.

Protocol:

1. Created mastermix for PCR reactions for a total of 64 wells. The following table shows volumes needed for 1 pCCR reaction, then volumes needed for a mastermix for 64 reactions:

	per reaction	7/31/19
# Samples	1	30
# Reactions	1.0	62
Template (RNA sample) (uL)	1.0	62.0
Sso Fast (uL)	10.0	682.0
Pf, 10 uM (uL)	0.5	34.1
Pr, 10 uM (uL)	0.5	34.1
DEPC-treated water (uL)	8.0	545.6
Total Volume in mastermix (uL)	20.0	1,357.8

Following Sam’s lab notebook entry, I used elongation factor primers to check for DNA contamination:

• EF1_qPCR_5’ (SRID 309) (Forward primer, Pf, SRID = 310)
• EF1_qPCR_3’ (SRID 310) (Reverse primer, Pr, SRID = 309)

These primer stocks are stored in a small fridge in FTR 213, and can be found using the primer database. The stock concentrations are 100 micromolar. I need to use a working stock at 10 uM, so I melted the stocks and diluted 15 uL of each stock in 135 uL DEPC-treated ultrapure water (150 uL total volume).

The total volume per reaction is 20 uL. After creating the mastermix, I pipetted 19 uL of mastermix into a qPCR well plate. NOTE: the type of plate is specific - it’s a white plate, and “low profile” - which is specified on the qPCR software. I then pipetted 1 uL of each sample (i.e. template), in duplicate, to the well plate. I loaded samples horizontally (A1, A2, A3 … etc.) for ease of reading data downstream. In addition to including a control sample, which has been processed alongside the other samples since homogenization (sample 571), I included a No Template Control (NTC, 1uL water added instead of a sample), and DNA isolated from Oly larvae back in March 2018 (sample 69a, RNA sample 8a) as a positive control. To seal the plate I used the clear tape-like cover, rather than the clear plastic caps. I did not vortex the well plate prior to the qPCR reaction.

I carried the well plate over to the qPCR room, loaded it onto the CFX Connect, and opened the MAESTRO software on the adjacent computer. I used the Wizard to help configure the run. Here are the steps to execute the run:

Select “User Defined”

Select Protocol: “CFX_2StepAmp_EVAGreen+Melt.prcl”

Select plate file: “QuickPlate_96 wells_sybr_white.pltd” - this ensures that all wells are measured. We don’t assign sample names prior to running, but can edit the data file after completion.

Select “Next”

Select “Save” - it will automatically save the file to Owl and filename will include the run date.

This is a screenshot immediately upon protocol initiation

I downloaded MAESTRO to my computer (Mac version), and edited the plate setup to include sample names, and color coded melt curves by sample type: GREEN is positive control (n=2); RED is NTC (n=1), and PINK is the homogenization/isolation/DNase control (n=1); BLUE are the samples.

Data and report are saved on github in the O.lurida_Stress repo.

The melt curve doesn’t look like Sam’s recent run. However, I realize that the primers were not O. lurida, but were C. gigas. I didn’t think to ask whether the primers needed to be O. lurida specific, but I’m guessing yes. I will plan to move forward with the next batch of Turbo DNase-ing, and will figure out which primers are optimal. Interestingly I did see some DNA, and a melt temperature, for the positive controls, but the fluorescence was not as high as Sam’s example. Also interesting is that my homogenization/isolation control (pink) had a weird peak, suggesting some contamination.

Here ares ome qPCR notes from Sam’s instructions:

• Keep RNA on ice while working with them, and store in -80 always.
• There are 2x SsoFast aliquots in the fridge, and also in the freezer in the “PCR supplies” box in the -20 (both in FTR 209).
• Mastermixes should be used the same day they are prepared, but can sit on ice for a few hours.
• qPCR plates can be prepared, then sealed and held in the fridge for a bit. For example, I could prepare one qPCR plate, then while it is running I can prepare another and hold it in the fridge until the machine is ready again.
• Always use the button to open/close the BioRAD CFX Connect lid - don’t manually close the lid

Quantified DNased RNA

Used Qubit HS RNA to measure RNA concentration in DNased samples. Approximate volume remaining for DNased RNA is 50 uL. I find it odd that some of my samples have more concentrated RNA after the DNasing. I will look in to that.

Date larvae collected	Cohort	Treatment	TISSUE SAMPLE #	Homo./RNA TUBE #	VOL RNAzol (mL)	MASS TISSUE (mg)	[RNA] ng/uL	Volume for DNase treatment	Amount of RNA in Dnase treatment (ug), max is 10 ug	Date Turbo Dnase treatment	[RNA] after Turbo Dnase treatment
5/24/17	Dabob Bay	10 Ambient	14-A	401	1	100	52.0	50	2.60	7/31/19	93.4
5/31/17	Dabob Bay	10 Ambient	31-A	402	1	10	140.0	50	7.00	7/31/19	114.0
5/26/17	Dabob Bay	10 Low	23-A	411	1	10	57.2	50	2.86	7/31/19	72.6
5/27/17	Dabob Bay	10 Low	27-A	412	1	10	60.8	50	3.04	7/31/19	31.2
6/12/17	Dabob Bay	6 Ambient	59-A	421	1	10	43.0	50	2.15	7/31/19	57.6
6/7/17	Dabob Bay	6 Low	51-A	431b	1	20	61.2	50	3.06	7/31/19	83.0
6/17/17	Dabob Bay	6 Low	72-A	432	1	50	47.6	50	2.38	7/31/19	74.0
5/25/17	Fidalgo Bay	10 Ambient	20-A	441	1	70	46.0	50	2.30	7/31/19	16.2
6/3/17	Fidalgo Bay	10 Ambient	38-A	442b	1	80	56.2	50	2.81	7/31/19	69.8
5/24/17	Fidalgo Bay	10 Low	16-A	451	1	70	68.4	50	3.42	7/31/19	68.4
5/24/17	Fidalgo Bay	10 Low	18-A	452b	1	80	48.4	50	2.42	7/31/19	97.2
5/26/17	Fidalgo Bay	6 Ambient	22-A	461b	1	100	54.0	50	2.70	7/31/19	84.0
5/29/17	Fidalgo Bay	6 Ambient	29-A	462b	1	60	69.8	50	3.49	7/31/19	106.0
5/25/17	Fidalgo Bay	6 Low	19-A	471b	1	100	71.0	50	3.55	7/31/19	108.0
5/26/17	Fidalgo Bay	6 Low	21-A	472b	1	70	64.0	50	3.20	7/31/19	97.0
5/20/17	Oyster Bay C1	10 Ambient	02-A	481	1	40	64.4	50	3.22	7/31/19	89.2
5/20/17	Oyster Bay C1	10 Ambient	04-A	482	1	60	67.2	50	3.36	7/31/19	22.2
5/23/17	Oyster Bay C1	10 Ambient	09-A	484	1	40	66.2	50	3.31	7/31/19	58.4
6/15/17	Oyster Bay C1	10 Ambient	66-A	491	1	20	126.0	50	6.30	7/31/19	58.4
6/14/17	Oyster Bay C1	10 Low	62-A	506	1	80	63.8	50	3.19	7/31/19	29.2
6/5/17	Oyster Bay C1	6 Ambient	45-A	513		30	156.0	50	7.80	7/31/19	142.0
5/21/17	Oyster Bay C1	6 Low	01-A	521	1	70	54.4	50	2.72	7/31/19	66.6
5/22/17	Oyster Bay C1	6 Low	07-A	522	1	20	60.8	50	3.04	7/31/19	32.2
6/15/17	Oyster Bay C1	6 Low	68-A	528	1	30	162.0	50	8.10	7/31/19	87.6
5/24/17	Oyster Bay C2	10 Ambient	17-A	531	1	60	88.2	50	4.41	7/31/19	95.4
5/23/17	Oyster Bay C2	10 Low	12-A	541	1	40	45.6	50	2.28	7/31/19	44.4
5/24/17	Oyster Bay C2	10 Low	13-A	542	1	30	82.0	50	4.10	7/31/19	32.8
6/3/17	Oyster Bay C2	6 Ambient	41-A	552b	1	80	64.8	50	3.24	7/31/19	74.6
5/21/17	Oyster Bay C2	6 Low	05-A	561	1	40	43.4	50	2.17	7/31/19	28.0
NA	RNA Control	RNA Control		571	1	10	LOW	50	LOW	7/31/19	LOW