DNasing RNA, Round I
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.
- 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:
|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 “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|