TITLE: Total RNA and DNA in microalgae: The extraction and quantification
AUTHORS: Yingyu Hu, Zoe V Finkel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This assay protocol for total RNA and DNA is adapted from Berdalet E. et al. and Liefer J.D. et al.</div><div class = "text-block">RNA and DNA are extracted from microalgae samples and then quantified by fluorochrome SYBR Green II. </div></div>

[STEPS]
?. [RNA and DNA extraction]
Freeze dry samples.
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							Freeze-drying should be as short as possible to reduce sample degradation.
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?. [RNA and DNA extraction]
Expose biosafety cabinet to UV light for  and clean working surface with decontamination solution.
?. [RNA and DNA extraction]
Prepare falcon tubes and tube rack in biosafety cabinet
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							  ABC1NumberTube typeUsage225 mL falcon tubesEDTA20% sarcosine3315 mL falcon tubes1 M Tris, 5 mM Tris, 1% STEB
ABC1NumberTube typeUsage225 mL falcon tubesEDTA20% sarcosine3315 mL falcon tubes1 M Tris, 5 mM Tris, 1% STEB
?. [RNA and DNA extraction]
Prepare Tris buffer
?. [RNA and DNA extraction]
Pour  Tris into an RNase free 15 mL Falcon tube
?. [RNA and DNA extraction]
Add   Tris into 500 mL RNase free water in the original package.
2.5 mL
?. [RNA and DNA extraction]
Prepare STEB ( )Require 500 uL for each sample and 500 uL for preparing 0.05% STEB in assay.
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?. [RNA and DNA extraction]
Prepare ice bath
?. [RNA and DNA extraction]
Pour sarcosine ( ) into an RNase free 5 mL falcon tube.
?. [RNA and DNA extraction]
Pour EDTA ( ) into an RNase free 5 mL falcon tube.
?. [RNA and DNA extraction]
Pour Tris buffer ( ,)  into an RNase free 15 mL falcon tube.
?. [RNA and DNA extraction]
Mix  sarcosine ( ) ,  EDTA ( )  and + Tris buffer ( ,)  to obtain STEB ( ).
500 µl
10 µl
9 mL
490 µl
?. [RNA and DNA extraction]
Remove samples from -80ºC freezer and place  .
on ice
?. [RNA and DNA extraction]
Add  Tris buffer ( ,) and  STEB ( ) to the bead tube. Place tubes .
500 µl
500 µl
on ice
?. [RNA and DNA extraction]
Transfer sample/blank filter to the tube.Forcepts must be cleaned by  ethanol.Blank filter should be the same type as the one with samples collected.
?. [RNA and DNA extraction]
Vortex immediately then put back .
on ice
?. [RNA and DNA extraction]
Disrupt samples on the bead mill at 6.5 m/s.
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?. [RNA and DNA extraction]
Keep tubes. Check the label on each tube, restore the label if it fades.
on ice
?. [RNA and DNA extraction]
Disrupt samples on the bead mill at 6.5 m/s.
?. [RNA and DNA extraction]
Keep tubes. Check the label on each tube, restore the label if it fades.
on ice
?. [RNA and DNA extraction]
Disrupt samples on the bead mill at 6.5 m/s
?. [RNA and DNA extraction]
Keep tubes. Check the label on each tube, restore the label if it fades.
on ice
?. [RNA and DNA extraction]
Disrupt samples on the bead mill at 6.5 m/s.
?. [RNA and DNA extraction]
Continuously shake homogenate in a multi-head vortex at the highest reasonable speed for
0 Room temperature
?. [RNA and DNA extraction]
In the biosafety cabinet, aliquot 150 uL homogenates into RNase free 600 uL tubes.
?. [RNA and DNA extraction]
Store at .
-80 °C
?. [The assay with nucleases]
Prepare ice bath.
?. [Fluorescence reading]
Prepare SYBR Green II working solution (SG-II WS)
?. [Standard curve and calculation]
RNA standard curve
?. [The assay with nucleases]
Expose biosafety cabinet to UV light for  and clean working surface with decontamination solution.
?. [The assay with nucleases]
Prepare falcon tubes, microtubes and tube racks in biosafety cabinet
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							* Maximum number of samples (including blanks) per assay is 20.
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?. [The assay with nucleases]
Organize and label the tubes as shown belowSet 1: Sample dilutionSet 2: StandardsSet 3: Sample reaction
?. [The assay with nucleases]
Label tubes for reagents as following. Add Tris buffer ( ,) to the reagent tubes:
?. [The assay with nucleases]
Add  Tris buffer ( ,) to each tube in Set 1
900 µl
?. [The assay with nucleases]
Add Tris buffer ( ,) to the tubes in Set 2. The unit of volume is uL.
?. [The assay with nucleases]
Add Tris buffer ( ,) to tubes in Set 3. The unit of volume is uL.
?. [Fluorescence reading]
After incubation, stop reaction by vortexing each tube for  and then place into the fridge.
?. [The assay with nucleases]
Add  STEB ( ) to Set 2.
250 µl
?. [The assay with nucleases]
Prepare RNase working solution Add  RNase primary stock () to RNase tube
10 µl
?. [Prepare standard and nucleases]
RNA primary standard ( )
?. [Prepare standard and nucleases]
Add  Tris buffer ( ,) to the original package and vortex.
1 mL
?. [Prepare standard and nucleases]
Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at .
-80 °C
?. [Prepare standard and nucleases]
DNA primary standard ()
?. [Prepare standard and nucleases]
RNase primary stock ( )
?. [Prepare standard and nucleases]
DNase primary stock ( = 10,000 U/mL)
?. [Prepare standard and nucleases]
Add  Tris buffer ( ,) to the original package and vortex.
2 mL
?. [Prepare standard and nucleases]
Aliquot 10 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at .
-80 °C
?. [Prepare standard and nucleases]
Add  Tris buffer ( ,) to the original package and vortex.
5 mL
?. [Prepare standard and nucleases]
Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at .
-20 °C
?. [Prepare standard and nucleases]
Add  Tris buffer ( ,) to the original package and vortex.
1 mL
?. [Prepare standard and nucleases]
Aliquot 100 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at .
-20 °C
?. [The assay with nucleases]
Place RNase, DNase primary stock, RNA primary standard, DNA primary standard and samples .
on ice
?. [The assay with nucleases]
Prepare  MgCl2
?. [The assay with nucleases]
Prepare    CaCl2
?. [The assay with nucleases]
Pour  MgCl2 solution into 5 mL RNase free Falcon tube
?. [The assay with nucleases]
Transfer  MgCl2 solution into 900 mM MgCl2 tube
180 µl
?. [The assay with nucleases]
Pour  CaCl2 solution into 5 mL RNase free Falcon tube
?. [The assay with nucleases]
Transfer  CaCl2 solution into 900 mM CaCl2 tube
180 µl
?. [The assay with nucleases]
Add  DNase primary stock (  ) to WS-A, keep WS-A .
60 µl
on ice
?. [The assay with nucleases]
Add  MgCl2 to WS-A and WS-B
60 µl
?. [The assay with nucleases]
Add   CaCl2 to WS-A and WS-B
60 µl
?. [The assay with nucleases]
Add   RNase to WS-B and WS-C, keep WS-B and WS-C  .
60 µl
on ice
?. [The assay with nucleases]
Turn on refrigerated centrifuge and set the temperature to .
4 °C
?. [The assay with nucleases]
Turn on shaker/incubator and set temperature to .
37 °C
?. [The assay with nucleases]
Centrifuge extracted samples
Centrifuge: 10000 34, 4 min, 4 10
?. [The assay with nucleases]
Prepare RNA secondary standardAdd  RNA primary standard to RNA standard tube and vortex. Keep  .
15 µl
on ice
?. [The assay with nucleases]
Prepare DNA secondary standard Add  DNA primary standard to DNA standard tube and vortex. Keep .
2 µl
on ice
?. [The assay with nucleases]
Load  WS-A to Tubes A in Set 2 and Set 3.
50 µl
?. [The assay with nucleases]
Load  WS-A to Tubes C in Set 2 and Set 3.
50 µl
?. [The assay with nucleases]
Load  WS-B to Tubes B in Set 2 and Set 3.
50 µl
?. [The assay with nucleases]
Load  WS-C to Tubes C in Set 2 and Set 3.
50 µl
?. [The assay with nucleases]
Add  centrifuged samples to its corresponding tubes in Set 1. Vortex each tube.
100 µl
?. [The assay with nucleases]
From Set 1, transfer of diluted samples to each corresponding tubes in Set 3.
250 µl
?. [The assay with nucleases]
Add RNA and DNA secondary standards to Set 2 tubes. The unit of volume is uL.
?. [The assay with nucleases]
Vortex each tube for  and place all tubes into the shaker/incubator at , continuously shaking at 200 RPM for .
37 °C
?. [Fluorescence reading]
Each 96-well microplate requires 1 mL of SG-II WS.
?. [Fluorescence reading]
Wrap 5 mL tube with foil, add  SYBR Green II 10,000X concentrate to each one mL Tris buffer ( ,) in biosafety cabinet.
7 µl
?. [Fluorescence reading]
Load  SG-II WS to each well in the microplate with 0.5 mL tip of stepper.Adhere black film on the top of a microplate lid and cover the plate with this lid.
10 µl
?. [Fluorescence reading]
Allow samples to reach  for  before loading the microplate.
0 Room temperature
?. [Fluorescence reading]
Load  working sample to the microplate. Standards must be included in each microplate.
190 µl
?. [Fluorescence reading]
Shake black film covered microplate for
37 Room temperature
?. [Fluorescence reading]
Setup microplate reader:
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							Plate: Greiner F bottom chimney well PP 96 well;
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							Endpoint reading: Ex 490 nm/Em 520 nm;
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							Shake: Continuous 5s at 600 rpm
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?. [Fluorescence reading]
Read fluorescence and export data to excel sheet.
?. [Standard curve and calculation]
DNA standard curve
?. [Standard curve and calculation]
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.
?. [Standard curve and calculation]
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.
?. [Standard curve and calculation]
Concentrations of RNA standards in the microplate
?. [Standard curve and calculation]
Calculate ρ
?. [Standard curve and calculation]
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
?. [Standard curve and calculation]
Concentrations of DNA standards in the microplate
?. [Standard curve and calculation]
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
?. [Standard curve and calculation]
Calculate δ
?. [Standard curve and calculation]
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
?. [The assay with nucleases]
Prepare STEB ( )
?. [The assay with nucleases]
Add  STEB ( ) to 0.05% STEB tube, and vortex.
500 µl