TITLE: Total RNA and DNA in Microalgae
AUTHORS: Yingyu Hu, Zoe V Finkel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Here we describe a protocol for extracting and quantifying bulk RNA and DNA from microalgae, which is adapted from Berdalet E. et al. (2005).</div><br/><div class = "text-block">RNA and DNA are extracted from microalgae samples and then quantified by fluorochrome SYBR Green II. </div><br/><br/><br/><br/><br/></div>

[STEPS]
?. [Day 1: Freeze-dry samples]
Freeze dry samples and blank filters. Freeze at   until processed.
-80 °C
Freeze-drying should be as short as possible to reduce sample degradation.The exact duration of freeze-drying depends on size of filter, quantity of sample and the size of container.
?. [Day 1: Prepare primary solutions]
Turn on UV light in biosafety cabinet for  and clean working surface with decontamination solution.
?. [Day 1: Prepare primary solutions]
Prepare Tris buffer
?. [Day 1: Prepare primary solutions]
Pour  Tris into an RNase free 15 mL Falcon tube
?. [Day 1: Prepare primary solutions]
Directly add   Tris into 500 mL RNase free water in its original package.
2.5 mL
?. [Day 1: Prepare primary solutions]
RNA primary standard solution ( )
?. [Day 1: Prepare primary solutions]
In the original package, the E. Coli Total RNA is of 1 mg/mL, in which total RNA is 200 ug.
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/sp_7940.pdf
?. [Day 1: Prepare primary solutions]
Uncap the original package of E. Coli Total RNA and directly add  Tris buffer ( ,) .Cap the package and vortex for a thorough mix.
800 µl
?. [Day 1: Prepare primary solutions]
Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at .
-80 °C
?. [Day 1: Prepare primary solutions]
DNA primary standard solution (≈ )
?. [Day 1: Prepare primary solutions]
Uncap the original package of Deoxyribonucleic acid from calf thymus and add    Tris buffer ( ,).
2 mL
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/d4522pis.pdf
?. [Day 1: Prepare primary solutions]
Cap the package. Do not vortex or sonicate.
?. [Day 1: Prepare primary solutions]
Keep the solution at  ~ overnight to completely solubilize the DNA. Gentle reversion is recommended.
0 °C
4 °C
?. [Day 1: Prepare primary solutions]
Aliquot 10 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at .
-80 °C
?. [Day 1: Prepare primary solutions]
Dilute 5 ul primary DNA standard solution with 95 ul Tris buffer ( ,) in a microtube (600 ul).Measure DNA concentration by using μdrop plate (sample volume: 4 ul)Use Tris buffer ( ,) as blank.
The dilution is to avoid saturated observation at 260 nm.
?. [Day 1: Prepare primary solutions]
DNA concentration (μg/ml) = (Abs260-Abs260 (blank))x 50 μg/ml x (10mm/0.5 mm) X DFWhere, DF=20.
If the measured DNA concentration is not close to  , check reverse pipetting technique.
?. [Day 1: Prepare primary solutions]
RNase primary stock solution ( )
?. [Day 1: Prepare primary solutions]
Uncap the original package of Ribonuclease A from bovin pancreas and add   Tris buffer ( ,). Cap the package and vortex for a thorough mix.
5 mL
?. [Day 1: Prepare primary solutions]
Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at .
-20 °C
?. [Day 1: Prepare primary solutions]
DNase primary stock solution ( = 10,000 U/mL)
?. [Day 1: Prepare primary solutions]
Uncap the original package of Deoxyribonuclease1 and add   Tris buffer ( ,) .Cap the package and vortex for a thorough mix.
1 mL
?. [Day 1: Prepare primary solutions]
Aliquot 100 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at .
-20 °C
?. [Day 2: Exact RNA and DNA]
Turn on UV light in biosafety cabinet for  and clean working surface with decontamination solution.
?. [Day 2: Exact RNA and DNA]
Prepare falcon tubes and tube rack in biosafety cabinet  AB1Volume of tube (mL)Contents in the tube250.5 M EDTA3520% sarcosine4151 M Tris5155 mM Tris6151% STEB
AB1Volume of tube (mL)Contents in the tube250.5 M EDTA3520% sarcosine4151 M Tris5155 mM Tris6151% STEB
?. [Day 2: Exact RNA and DNA]
Prepare STEB ( )
Use the following formula to determine the total volume of 1% STEB required:(# samples + # blank filters) X (500 ul) + (500 ul) = total volume of 1% STEB required
?. [Day 2: Exact RNA and DNA]
Pour sarcosine ( ) into an RNase free 5 mL falcon tube.
?. [Day 2: Exact RNA and DNA]
Pour EDTA ( ) into an RNase free 5 mL falcon tube.
?. [Day 2: Exact RNA and DNA]
Pour Tris buffer ( ,)  into an RNase free 15 mL falcon tube.
?. [Day 2: Exact RNA and DNA]
Mix  sarcosine ( ) ,  EDTA ( )  and + Tris buffer ( ,)  to obtain STEB ( ).
500 µl
10 µl
9 mL
490 µl
?. [Day 2: Exact RNA and DNA]
Prepare ice bath
?. [Day 2: Exact RNA and DNA]
Remove freeze-dried samples from -80ºC freezer and place them .
on ice
?. [Day 2: Exact RNA and DNA]
Add  Tris buffer ( ,) and  STEB ( ) to the bead tube. Place tubes .
500 µl
500 µl
on ice
?. [Day 2: Exact RNA and DNA]
Rinse forceps by   ethanol and air dry.
?. [Day 2: Exact RNA and DNA]
Transfer sample/blank filter into the bead tube by using clean forceps.
?. [Day 2: Exact RNA and DNA]
Vortex immediately then put back .
on ice
?. [Day 2: Exact RNA and DNA]
Disrupt samples on the bead mill at 6.5 m/s.
?. [Day 2: Exact RNA and DNA]
Keep tubes. Check the label on each tube, restore the label if it fades.
on ice
?. [Day 2: Exact RNA and DNA]
Disrupt samples on the bead mill at 6.5 m/s.
?. [Day 2: Exact RNA and DNA]
Keep tubes. Check the label on each tube, restore the label if it fades.
on ice
?. [Day 2: Exact RNA and DNA]
Disrupt samples on the bead mill at 6.5 m/s
?. [Day 2: Exact RNA and DNA]
Keep tubes. Check the label on each tube, restore the label if it fades.
on ice
?. [Day 2: Exact RNA and DNA]
Disrupt samples on the bead mill at 6.5 m/s.
?. [Day 2: Exact RNA and DNA]
Continuously shake homogenate in a multi-head vortex at the highest speed for
0 Room temperature
Votex mixer should be able to remain stable on the bench under this vortex speed.
?. [Day 2: Exact RNA and DNA]
In the biosafety cabinet, aliquot 150 uL of homogenates into RNase free 600 uL tubes.
?. [Day 2: Exact RNA and DNA]
Freeze at  until analyzed.
-80 °C
?. [Day 3: Run the assay ]
Prepare ice bath.
?. [Day 3: Run the assay ]
Turn on UV light in biosafety cabinet for  and clean working surface with decontamination solution.
?. [Day 3: Run the assay ]
Prepare falcon tubes, microtubes and tube racks in biosafety cabinet* Maximum number of samples (including blanks) per assay is 20.  ABC1Number of tubesType of tubesContents265 mL falcon tubes1 M MgCl2, 1 M CaCl2, Working solution A (WS-A), Working solution B (WS-B), Working solution C (WS-C), Sybr Green II working solution (SG-II WS)3215 mL falcon tubes5 mM Tris buffer, 0.05% STEB43600 uL RNase free tubesRNase working solution, 900 mM MgCl2, 900 mM CaCl2521.7 mL RNase free tubesSecondary RNA standard solution, Secondary DNA standard solution6331.7 mL RNase free tubesRNA standard solutions for RNA standard curves, DNA standard soutions for DNA standard curves7N= total number of samples and blanks1.7 mL RNase free tubesSamples and blanks83XN1.7 mL RNase free tubesDiluted samples and blanks95Microtube racksTubes of 600 uL and 1.7 mL101Tube racksFalcon tubes
ABC1Number of tubesType of tubesContents265 mL falcon tubes1 M MgCl2, 1 M CaCl2, Working solution A (WS-A), Working solution B (WS-B), Working solution C (WS-C), Sybr Green II working solution (SG-II WS)3215 mL falcon tubes5 mM Tris buffer, 0.05% STEB43600 uL RNase free tubesRNase working solution, 900 mM MgCl2, 900 mM CaCl2521.7 mL RNase free tubesSecondary RNA standard solution, Secondary DNA standard solution6331.7 mL RNase free tubesRNA standard solutions for RNA standard curves, DNA standard soutions for DNA standard curves7N= total number of samples and blanks1.7 mL RNase free tubesSamples and blanks83XN1.7 mL RNase free tubesDiluted samples and blanks95Microtube racksTubes of 600 uL and 1.7 mL101Tube racksFalcon tubes
?. [Day 3: Run the assay ]
Organize and label the tubes as shown belowSet 1: In microtube rack, label 1.7 mL tubes for samples and blanks to be further diluted.Set 2:In microtube rack, label 1.7 mL tubes for RNA (marked in pink) and DNA (marked in blue) standard solutions to be used as standard curves.Tubes A is for standard solutions treated with working solution A (WS-A)Tubes B is for standard solutions treated with working solution B (WS-B)Tubes C is for standard solutions treated with working solution C (WS-C)Set 3:In microtube rack, label 1.7 mL tubes for diluted samples and blanks.Tubes A is for diluted samples and blanks treated with working solution A (WS-A)Tubes B is for diluted samples and blanks treated with working solution B (WS-B)Tubes C is for diluted samples and blanks treated with working solution C (WS-C)
?. [Day 3: Run the assay ]
Label tubes for reagents as following. Follow the sheet, add Tris buffer ( ,) to the reagent tubes:
?. [Day 3: Run the assay ]
Add  Tris buffer ( ,) to each tube in Set 1
900 µl
?. [Day 3: Run the assay ]
Follow the sheet, add Tris buffer ( ,) to each tube in Set 2. The unit of volume is uL.
?. [Day 3: Run the assay ]
Follow the sheet, add Tris buffer ( ,) to each tube in Set 3. The unit of volume is uL.
?. [Day 3: Run the assay ]
Prepare STEB ( ) Add  STEB ( ) to 0.05% STEB tube, and vortex.
500 µl
?. [Day 3: Run the assay ]
Add  STEB ( ) to each tube in Set 2.
250 µl
?. [Day 3: Run the assay ]
Place RNase and DNase primary stock solutions, RNA and DNA primary standard solutions and samples .
on ice
?. [Day 3: Run the assay ]
Turn on refrigerated centrifuge and set the temperature to .
4 °C
?. [Day 3: Run the assay ]
Turn on shaker/incubator and set temperature to .
37 °C
?. [Day 3: Run the assay ]
Prepare  MgCl2
?. [Day 3: Run the assay ]
Pour  MgCl2 solution into 5 mL RNase free Falcon tube
?. [Day 3: Run the assay ]
Transfer  MgCl2 solution into 900 mM MgCl2 tube
180 µl
?. [Day 3: Run the assay ]
Add  MgCl2 to WS-A and WS-B
60 µl
?. [Day 3: Run the assay ]
Prepare    CaCl2
?. [Day 3: Run the assay ]
Pour  CaCl2 solution into 5 mL RNase free Falcon tube
?. [Day 3: Run the assay ]
Transfer  CaCl2 solution into 900 mM CaCl2 tube
180 µl
?. [Day 3: Run the assay ]
Add   CaCl2 to WS-A and WS-B
60 µl
?. [Day 3: Run the assay ]
Prepare RNase working solution Add  RNase primary stock solution () to RNase tube
10 µl
?. [Day 3: Run the assay ]
Add   RNase to WS-B and WS-C.Keep WS-B and WS-C  .
60 µl
on ice
?. [Day 3: Run the assay ]
Add  DNase primary stock solution (  ) to WS-A.Keep WS-A .
60 µl
on ice
?. [Day 3: Run the assay ]
Centrifuge extracted samples
Centrifuge: 10000 34, 4 min, 4 10
?. [Day 3: Run the assay ]
Prepare RNA secondary standard solution Add  RNA primary standard solution to RNA standard tube and vortex. Keep  .
15 µl
on ice
?. [Day 3: Run the assay ]
Prepare DNA secondary standard solution Add  DNA primary standard solution to DNA standard tube and vortex. Keep .
2 µl
on ice
?. [Day 3: Run the assay ]
Load  WS-A to Tubes A in Set 2 and Set 3.
50 µl
?. [Day 3: Run the assay ]
Load  WS-A to Tubes C in Set 2 and Set 3.
50 µl
?. [Day 3: Run the assay ]
Load  WS-B to Tubes B in Set 2 and Set 3.
50 µl
?. [Day 3: Run the assay ]
Load  WS-C to Tubes C in Set 2 and Set 3.
50 µl
?. [Day 3: Run the assay ]
Add  centrifuged samples to its corresponding tubes in Set 1. Vortex each tube.
100 µl
?. [Day 3: Run the assay ]
From Set 1, transfer  of diluted samples to each corresponding tubes in Set 3.
250 µl
?. [Day 3: Run the assay ]
Follow the sheet:Add RNA secondary standard to tubes (marked in pink) in Set 2.Add  DNA secondary standard to tubes (marked in blue) in Set 2. The unit of volume is uL.
?. [Day 3: Run the assay ]
Vortex each tube for  and place all tubes into the shaker/incubator at , continuously shaking at 200 RPM for .
37 °C
?. [Read fluorescence]
Prepare SYBR Green II working solution (SG-II WS)
?. [Read fluorescence]
Each 96-well microplate requires 1 mL of SG-II WS.
?. [Read fluorescence]
Wrap 5 mL tube with foil, add  SYBR Green II 10,000X concentrate to each one mL Tris buffer ( ,) in biosafety cabinet.
7 µl
?. [Read fluorescence]
Load  SG-II WS to each well in the microplate with 0.5 mL tip of stepper.
10 µl
Wipe or dab the liquid drop on the outside of the tip, avoid wiping the tip open before dispensing the liquid.
?. [Read fluorescence]
Adhere black film on the top of a microplate lid and cover the plate with this lid.
?. [Read fluorescence]
After incubation, vortex each tube for  and then place into the fridge to stop the reaction.
?. [Read fluorescence]
Allow samples to reach  for  before loading the microplate.
0 Room temperature
?. [Read fluorescence]
Load  working sample to the microplate. Standards must be included in each microplate.Pink area: RNA standard solutions for RNA standard curvesBlue area: DNA standard solutions for DNA standard curvesYellow area: Samples and blanks
190 µl
Wipe or dab the liquid drop on the outside of the tip, avoid wiping the tip open before dispensing the liquid.
?. [Read fluorescence]
Shake black film covered microplate at  for
37 Room temperature
?. [Read fluorescence]
Setup microplate reader:Plate: Greiner F bottom chimney well PP 96 well;Shake: Continuous 5s at 600 rpmEndpoint reading: Ex 490 nm/Em 520 nm;
?. [Read fluorescence]
Read fluorescence and export data to excel sheet.
?. [Calculate  ]
RNA standard curve
?. [Calculate  ]
Concentrations of RNA standards in the microplate
?. [Calculate  ]
Slope of fluorescence in Tube A vs concentration of RNA standard gives mRNA+DNase Slope of fluorescence in Tube B vs concentration of RNA standard gives mRNA+RNase
?. [Calculate  ]
Calculate ρ
?. [Calculate  ]
Total RNA of the samplesWhere,RFUA and RFUC are the fluorescence in Tube A and Tube C of the same sample.RFUABlank and RFUCBlank are the fluorescence in Tube A and Tube C of the blank.
?. [Calculate  ]
DNA standard curve
?. [Calculate  ]
Concentrations of DNA standards in the microplate
?. [Calculate  ]
Slope of fluorescence in Tube A vs concentration of DNA standard gives mDNA+DNase Slope of fluorescence in Tube B vs concentration of DNA standard gives mDNA+RNase
?. [Calculate  ]
Calculate δ
?. [Calculate  ]
Total DNA of the samplesWhere,RFUB and RFUC are the fluorescence in Tube B and Tube C of the same sampleRFUBBlank and RFUCBlank are the fluorescence in Tube B and Tube C of the blank.
?. [Calculate  ]
Dilution factor=40If,Sample is extracted by 1 mL extraction reagentIn Set 1, sample is diluted to 1/10In Set 3, diluted by Tris and all working solutions to 250/950In microplate, diluted by SG-II WS to 190/200