Feb 20, 2023

Public workspaceTotal RNA and DNA from Microalgae (12 samples per microplate) V.11

DOI
dx.doi.org/10.17504/protocols.io.6qpvro85bvmk/v11
  • 1Dalhousie University
  • Marine Microbial Macroecology Lab
    Tech. support email: ruby.hu@dal.ca
Ying-Yu Hu
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DOI: dx.doi.org/10.17504/protocols.io.6qpvro85bvmk/v11
Protocol Citation: Ying-Yu Hu, Zoe V. Finkel 2023. Total RNA and DNA from Microalgae (12 samples per microplate) . protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvro85bvmk/v11Version created by Ying-Yu Hu
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 19, 2022
Last Modified: February 20, 2023
Protocol Integer ID: 71512
Keywords: RNA, DNA, SYBR Green II, DNase, RNase, microalgae, fluorescence
Funders Acknowledgements:
Simons Collaborative on Ocean Processes and Ecology
Grant ID: 723789
Abstract
Here we describe a protocol for extracting and quantifying bulk RNA and DNA from microalgae, which is adapted from Berdalet E. et al. (2005).

RNA and DNA are extracted from microalgae samples and then quantified by fluorochrome SYBR Green II.

The level of sensitivity of this method is set at ca. 20 ~300 ng RNA and 10 ~ 100 ng DNA (ml assay)-1.



CITATION
Berdalet E, Roldán C, Olivar MP, Lysnes K. Quantifying RNA and DNA in planktonic organisms with SYBR Green II and nucleases. Part A. Optimisation of the assay. Scientia Marina.
LINK
https://doi.org/10.3989/scimar.2005.69n11

CITATION
Berdalet E, Roldán C, Olivar MP. Quantifying RNA and DNA in planktonic organisms with SYBR Green II and nucleases. Part B. Quantification in natural samples. Scientia Marina.
LINK
https://doi.org/10.3989/scimar.2005.69n117







Guidelines
Estimation of RNA/DNA in the collected microalgae samples:

Under replete condition, RNA and DNA is about 5.7% and 1% in total dry mass, while Chl-a is bout 1.1% in total dry mass. Therefore, RNA_ug/L = Chl-a_ug/L X (5.7/1.1), DNA_ug/L = Chl-a_ug/L X (1/1.1).

Common dilution from sample collected on the filter to assay is 1/40.
Materials
STEP MATERIALS
ReagentRibonuclease A from bovine pancreasSigma AldrichCatalog #R6513-50MG
ReagentDEOXYRIBONUCLEASE1 RNase and Protease FreeBioshopCatalog # DRB002.10
ReagentMagnesium chloride solutionSigma AldrichCatalog #63069-100ML
ReagentCalcium chloride solutionSigma AldrichCatalog #21115-100ML
ReagentSYBR™ Green II RNA Gel Stain, 10,000X concentrate in DMSOThermo FisherCatalog #S7564
ReagentTris(hydroxymethyl)aminomethane hydrochloride 1M pH 8.0 RNase freeFisher ScientificCatalog #AAJ60080AK
ReagentDeoxyribonucleic acid from calf thymusSigma AldrichCatalog #D4522-1MG
ReagentN-Lauroylsarosine sodium salt solution (20% RNase/DNase free)Sigma AldrichCatalog #L744-50mL
ReagentEDTA buffer (0.5M DNase/RNase free)BioshopCatalog #EDT333.100
ReagentUltraPure™ DNase/RNase-Free Distilled WaterThermofisherCatalog #10977023
ReagentE. coli Total RNAThermo Fisher ScientificCatalog #AM7940

Protocol materials
ReagentCalcium chloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #21115-100ML
ReagentSYBR™ Green II RNA Gel Stain, 10,000X concentrate in DMSOThermo FisherCatalog #S7564
ReagentDeoxyribonucleic acid from calf thymusMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4522-1MG
ReagentN-Lauroylsarosine sodium salt solution (20% RNase/DNase free)Merck MilliporeSigma (Sigma-Aldrich)Catalog #L744-50mL
ReagentEDTA buffer (0.5M DNase/RNase free)BioshopCatalog #EDT333.100
ReagentUltraPure™ DNase/RNase-Free Distilled WaterThermofisherCatalog #10977023
ReagentRibonuclease A from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #R6513-50MG
ReagentDEOXYRIBONUCLEASE1 RNase and Protease FreeBioshopCatalog # DRB002.10
ReagentE. coli Total RNAThermo Fisher ScientificCatalog #AM7940
ReagentMagnesium chloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #63069-100ML
ReagentTris(hydroxymethyl)aminomethane hydrochloride 1M pH 8.0 RNase freeFisher ScientificCatalog #AAJ60080AK
ReagentTris(hydroxymethyl)aminomethane hydrochloride 1M pH 8.0 RNase freeFisher ScientificCatalog #AAJ60080AK
ReagentUltraPure™ DNase/RNase-Free Distilled WaterThermofisherCatalog #10977023
ReagentE. coli Total RNAThermo Fisher ScientificCatalog #AM7940
ReagentDeoxyribonucleic acid from calf thymusMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4522-1MG
ReagentRibonuclease A from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #R6513-50MG
ReagentDEOXYRIBONUCLEASE1 RNase and Protease FreeBioshopCatalog # DRB002.10
ReagentN-Lauroylsarosine sodium salt solution (20% RNase/DNase free)Merck MilliporeSigma (Sigma-Aldrich)Catalog #L744-50mL
ReagentEDTA buffer (0.5M DNase/RNase free)BioshopCatalog #EDT333.100
ReagentMagnesium chloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #63069-100ML
ReagentCalcium chloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #21115-100ML
ReagentSYBR™ Green II RNA Gel Stain, 10,000X concentrate in DMSOThermo FisherCatalog #S7564
Safety warnings
No data is available addressing the mutagenicity or toxicity of SYBR® Green II Nucleic Acid Gel Stain. Because this reagent binds to nucleic acids, it should be treated as a potential mutagen and used with appropriate care. The DMSO stock solution should be handled with particular caution as DMSO is known to facilitate the entry of organic molecules into tissues. We strongly recommend using double gloves when handling the DMSO stock solution. As with all nucleic acid stains, solutions of SYBR Green II Nucleic Acid Gel Stain should be poured through activated charcoal before disposal or collected in waste container to be treated later. The charcoal must then be incinerated to destroy the dye.
Day 1: Freeze-dry samples
Day 1: Freeze-dry samples
Freeze dry samples and blank filters. Freeze at Temperature-80 °C until processed.
Note
  1. Freeze-drying should be as short as possible to reduce sample degradation.
  2. The exact duration of freeze-drying depends on size of filter, quantity of sample and the size of container.

Equipment
FreeZone® 2.5 L Benchtop Freeze Dryers
NAME
Labconco®
BRAND
700202000
SKU

Day 1: Prepare primary solutions
Day 1: Prepare primary solutions
Turn on UV light in biosafety cabinet for Duration00:15:00
Clean working surface with decontamination solution.
Prepare Tris buffer Concentration5 mM Ph8.0
Pour Concentration1 M Ph8.0 Tris into an RNase free 15 mL Falcon tube
ReagentTris(hydroxymethyl)aminomethane hydrochloride 1M pH 8.0 RNase freeVWR InternationalCatalog #AAJ60080AK
Equipment
Falcon® Centrifuge Tubes
NAME
Polypropylene, Sterile, 15 mL
TYPE
Corning®
BRAND
352096
SKU

Directly add Amount2.5 mL Concentration1 M Ph8.0 Tris into 500 mL RNase free water in its original package.
ReagentUltraPure™ DNase/RNase-Free Distilled WaterVWR InternationalCatalog #10977023
Equipment
BT Barrier Pipet Tips
NAME
Pre-Sterile
TYPE
Neptune®
BRAND
BT1250, BT100, BT10
SKU

RNA primary standard solution (Concentration200 ug/ml )
In the original package, the frozen E. Coli Total RNA is of 1 mg/mL, in which total RNA is 200 ug.
ReagentE. coli Total RNAVWR InternationalCatalog #AM7940

Note

Uncap the original package of E. Coli Total RNA and directly add Amount800 µL Tris buffer (Concentration5 mM ,Ph8.0 ) .
Cap the package and vortex for a thorough mix.
Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at Temperature-80 °C
Equipment
Finnpipette Stepper Pipette
NAME
Thermo Scientific™
BRAND
4540000
SKU

Equipment
Finntip stepper pipette tips
NAME
500 ul (sterile)
TYPE
Thermo Scientific
BRAND
Thermo Scientific™ 9404173
SKU

DNA primary standard solution (≈ Concentration500 ug/ml )
Uncap the original package of Deoxyribonucleic acid from calf thymus and add Amount2 mL Tris buffer (Concentration5 mM ,Ph8.0 ).
ReagentDeoxyribonucleic acid from calf thymusVWR InternationalCatalog #D4522-1MG

Note

Cap the package. Do not vortex or sonicate.
Keep the solution at Temperature0 °C ~Temperature4 °C overnight to completely solubilize the DNA. Gentle reversion is recommended.
Aliquot 10 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at Temperature-80 °C
Equipment
Finntip stepper pipette tips
NAME
500 ul (sterile)
TYPE
Thermo Scientific
BRAND
Thermo Scientific™ 9404173
SKU

RNase primary stock solution (Concentration10 mg/ml )
Uncap the original package of Ribonuclease A from bovin pancreas and add Amount5 mL Tris buffer (Concentration5 mM ,Ph8.0 ).
Cap the package and vortex for a thorough mix.
ReagentRibonuclease A from bovine pancreasVWR InternationalCatalog #R6513-50MG
Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at Temperature-20 °C
Equipment
Finntip stepper pipette tips
NAME
500 ul (sterile)
TYPE
Thermo Scientific
BRAND
Thermo Scientific™ 9404173
SKU

Equipment
Finntip™ Stepper Pipette Tips
NAME
500 ul (Sterile)
TYPE
Thermo Scientific
BRAND
21-377-149
SKU

DNase primary stock solution (Concentration5 mg/ml = 10,000 U/mL)
Uncap the original package of Deoxyribonuclease1 and add Amount1 mL Tris buffer (Concentration5 mM ,Ph8.0 ) .
Cap the package and vortex for a thorough mix.
ReagentDEOXYRIBONUCLEASE1 RNase and Protease FreeVWR InternationalCatalog # DRB002.10

Aliquot 70 uL to 600 uL RNase free microtubes (every assay required 60 uL). Keep frozen at Temperature-20 °C .

Day 2: Exact RNA and DNA
Day 2: Exact RNA and DNA
Turn on UV light in biosafety cabinet for Duration00:15:00
Clean working surface with decontamination solution.
Prepare falcon tubes and tube rack in biosafety cabinet
Volume of the tube (mL)Content in the tube
50.5 M EDTA
520% sarcosine
505 mM Tris
15 or 501% STEB

Equipment
Falcon® Centrifuge Tubes
NAME
Polypropylene, Sterile, 15 mL
TYPE
Corning®
BRAND
352096
SKU

Equipment
Falcon® Centrifuge Tubes
NAME
Polypropylene, Sterile, 50 mL
TYPE
Corning®
BRAND
352070
SKU

Prepare STEB (Concentration1 % )
Note
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

Pour sarcosine (Concentration20 % ) into an RNase free 5 mL falcon tube.
ReagentN-Lauroylsarosine sodium salt solution (20% RNase/DNase free)VWR InternationalCatalog #L744-50mL
Pour EDTA (Concentration0.5 M ) into an RNase free 5 mL falcon tube.
ReagentEDTA buffer (0.5M DNase/RNase free)VWR InternationalCatalog #EDT333.100
Pour Tris buffer (Concentration5 mM ,Ph8.0 ) into an RNase free 50 mL falcon tube.
Mix Amount500 µL sarcosine (Concentration20 % ) , Amount10 µL EDTA (Concentration0.5 M ) and Amount9 mL +Amount490 µL Tris buffer (Concentration5 mM ,Ph8.0 ) to obtain STEB (Concentration1 % ).
Prepare ice bath
Remove freeze-dried samples from -80ºC freezer and place them TemperatureOn ice .
Add Amount500 µL Tris buffer (Concentration5 mM ,Ph8.0 ) and Amount500 µL STEB (Concentration1 % ) to the bead tube. Place tubes TemperatureOn ice .
Equipment
LYSING TUBES
NAME
MATRIX D 2 mL/15 mL
TYPE
MP BIOMEDICALS
BRAND
116913500/116933050
SKU

Rinse forceps by Concentration70 % volume ethanol and air dry.
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Millipore
BRAND
XX6200006P
SKU

Transfer sample/blank filter into the bead tube by using clean forceps.
Invert immediately then put back TemperatureOn ice .
20s
Disrupt samples on the bead mill at 6.5 m/s.
Equipment
Fastprep-24 5G™ Sample Preparation Instrument
NAME
MP Biomedicals
BRAND
116005500
SKU

30s
Keep tubes TemperatureOn ice . Check the label on each tube, restore the label if it fades.
1m 30s
Disrupt samples on the bead mill at 6.5 m/s.
30s
Keep tubes TemperatureOn ice . Check the label on each tube, restore the label if it fades.
1m 30s
Disrupt samples on the bead mill at 6.5 m/s
30s
Keep tubes TemperatureOn ice . Check the label on each tube, restore the label if it fades.
1m 30s
Disrupt samples on the bead mill at 6.5 m/s.
30s
Continuously shake homogenate in a multi-head vortex at the highest speed for Duration01:00:00 TemperatureRoom temperature
Note
Votex mixer should be able to remain stable on the bench under this vortex speed.

1h
In the biosafety cabinet, transfer all homogenate into RNase free 2 mL micro-tube.
Freeze at Temperature-80 °C until analyzed.
Day 3: Run the assay
Day 3: Run the assay
Prepare ice bath.
Turn on UV light in biosafety cabinet for Duration00:15:00
Clean working surface with decontamination solution.
Prepare falcon tubes, microtubes and tube racks in biosafety cabinet
Number of tubesType of tubesContents
55 mL falcon tubes1 M MgCl2
1 M CaCl2
Working solution A (WS-A)
Working solution B (WS-B)
Working solution C (WS-C)
150 mL falcon tube5 mM Tris buffer
115 mL falcon tubes0.05% STEB
62 mL RNase free tubesRNase working solution
RNA secondary standard solution
DNA tertiary standard solution
900 mM MgCl2
900 mM CaCl2
Sybr green working solution (SG-II WS)
1600 uL RNase free tubeDNA secondary standard
242 mL RNase free tubesRNA standard solutions for RNA standard curves
DNA standard solutions for DNA standard curves
N= total number of samples and blanks2 mL RNase free tubesSamples and blanks
3XN2 mL RNase free tubesDiluted samples and blanks
4Microtube racksTubes of 2 mL in Set 1
Tubes of 2 mL in Set A
Tubes of 2 mL in Set B
Tubes of 2 mL in Set C
1Tube racksFalcon tubes

Equipment
Screw-Cap Centrifuge Tube
NAME
5 mL
TYPE
VWR
BRAND
10002-738
SKU

Day 3: Run the assay (Caution: It is a long procedure!)
Day 3: Run the assay (Caution: It is a long procedure!)
Organize and label the tubes as shown below

Set 1:
In microtube rack, label 2 mL tubes for samples and blanks to be further diluted.

Set A, B and C:
In microtube rack, label 2 mL tubes for RNA (marked in pink), DNA (marked in blue) standard solutions and samples (marked in yellow)
Set A is for working solution A (WS-A) treatment, i.e. treated with DNase
Set B is for working solution B (WS-B) treatment, i.e. treated with RNase
Set C is for working solution A (WS-A) and C (WS-C) treatment, i.e. treated with DNase and RNase




Label tubes for reagents as following.
Follow the sheet, add Tris buffer (Concentration5 mM ,Ph8.0 ) to the reagent tubes:

Content5 mM Tris (uL)
SG-II WS1000+250
WS-A2X1000+820
WS-B2X1000+820
WS-C2X1000+940
RNase380
900 mM MgCl240
900 mM CaCl240
RNA secondary990+495
DNA secondary95
DNA tertiary 960
0.05% STEB9X1000 + 500


Thaw Sybr green II at room temperature
ReagentSYBR™ Green II RNA Gel Stain, 10,000X concentrate in DMSOVWR InternationalCatalog #S7564
Add Amount900 µL Tris buffer (Concentration5 mM ,Ph8.0 ) to each tube in Set 1

Note
Depending on the dilution of extracted sample

Follow the sheet, add Tris buffer (Concentration5 mM ,Ph8.0 ) to each tube in Set A, B and C. The unit of volume is uL.




Prepare STEB (Concentration0.05 % )
Add Amount500 µL STEB (Concentration1 % ) to 0.05% STEB tube, and vortex.
Add Amount250 µL STEB (Concentration0.05 % ) to RNA and DNA standards in Set A, B and C by reverse pipetting.

Place RNase and DNase primary stock solutions, RNA and DNA primary standard solutions and samples TemperatureOn ice .
Turn on refrigerated centrifuge and set the temperature to Temperature4 °C .
Equipment
CENTRIFUGE 5430 R
NAME
Eppendorf
BRAND
MP2231000510
SKU

Turn on shaker/incubator and set temperature to Temperature37 °C .
Equipment
SHAKING INCUBATOR
NAME
71L
TYPE
Corning® LSE™
BRAND
6753
SKU

Prepare Concentration900 mM MgCl2
Pour Concentration1 M MgCl2 solution into 5 mL RNase free Falcon tube
ReagentMagnesium chloride solutionVWR InternationalCatalog #63069-100ML

Transfer Amount360 µL Concentration1 M MgCl2 solution into 900 mM MgCl2 tube
Add Amount60 µL Concentration900 mM MgCl2 to WS-A
Add Amount60 µL Concentration900 mM MgCl2 to WS-B
Prepare Concentration900 mM CaCl2
Pour Concentration1 M CaCl2 solution into 5 mL RNase free Falcon tube
ReagentCalcium chloride solutionVWR InternationalCatalog #21115-100ML

Transfer Amount360 µL Concentration1 M CaCl2 solution into 900 mM CaCl2 tube
Add Amount60 µL Concentration900 mM CaCl2 to WS-A
Add Amount60 µL Concentration900 mM CaCl2 to WS-B
Prepare SG-II WS
Centrifuge one tube of SG-II concentrate at TemperatureRoom temperature Centrifigation13000 rpm, 00:05:00 to deposit DMSO.
Wrap SG-II WS tube with foil, transfer supernatant of SYBR Green II 10,000X concentrate to SG-II WS tube in biosafety cabinet ( Concentration8.75 ul per 1.25 mL Tris)
Note
Any step involving SYBR Green II should be operated in dark room or at least dim light.
Prepare Sybr green II WS at this step to allow enough time for stabilization.


Note
Lunch break!

Prepare RNase working solution Concentration0.5 mg/ml
Add Amount20 µL RNase primary stock solution (Concentration10 mg/ml ) to RNase tube

Add Amount60 µL Concentration0.5 mg/ml RNase to WS-B.
Keep WS-B TemperatureOn ice .
Add Amount60 µL Concentration0.5 mg/ml RNase to WS-C.
Keep WS-C TemperatureOn ice .
Add Amount60 µL DNase primary stock solution ( Concentration5 mg/ml ) to WS-A.
Keep WS-A TemperatureOn ice .
Centrifuge extracted samples Centrifigation10000 x g, 4°C, 00:04:00
Prepare RNA secondary standard solution Concentration2 ug/ml
Add Amount15 µL RNA primary standard solution to RNA standard tube and mix.
Keep TemperatureOn ice .
Prepare DNA secondary standard solution (Concentration25 ug/ml )
Add Amount5 µL primary DNA standard solution (Concentration500 ug/ml ) to DNA secondary tube and mix.

Note
DNA secondary standard will also be used to verify the actual concentration of DNA by using μdrop plate.

Note
Avoid vortexing DNA standard. Mix with pipette tip by aspiring up and down several times.

Prepare DNA tertiary standard solution Concentration1 ug/ml
Add Amount40 µL DNA secondary solution (Concentration25 ug/ml ) to DNA tertiary standard tube and mix.
Keep TemperatureOn ice .

Load Amount50 µL WS-A to tubes in Set A.
Note
From 59 to 62: Reverse pipetting
Decontaminate pipet between different WS.

Load Amount50 µL WS-A to tubes Set C.
Load Amount50 µL WS-B to tubes in Set B.
Load Amount50 µL WS-C to tubes in Set C.
Add Amount100 µL centrifuged samples to its corresponding tubes in Set 1.
Gently invert the tube to mix sample.
From Set 1, transfer Amount250 µL of diluted samples to each corresponding tubes (marked in yellow) in Set A, B and C.
Add RNA secondary standard to tubes (marked in pink) in Set A, B and C.
Add DNA secondary standard to tubes (marked in blue) in Set A, B and C.
The unit of volume is uL.

Note
In order to avoid cross contamination from RNase or DNase, use one tip for each dispensing.
Pipette solution in the tube up and down for mixing.


Invert each tube to mix well and place all tubes into the shaker/incubator at Temperature37 °C , continuously shaking at 200 RPM for Duration00:20:00 .
Note
Incubation time is critical. Temperature might be disturbed by door open/close. Don't start the timer until temperature returns to 37°C.

20m
After incubation, invert each tube for mixing and then place into the fridge to stop the reaction.
Day 3: Verify DNA concentration and SG-II absorbance
Day 3: Verify DNA concentration and SG-II absorbance
Measure DNA secondary concentration by using μdrop plate (sample volume: 4 ul)
Use Tris buffer (Concentration5 mM ,Ph8.0 ) as blank.
Equipment
µDrop™ Plates
NAME
Thermo Scientific
BRAND
N12391
SKU

Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU

DNA_primary concentration (μg/ml) = (Abs260-Abs260 (blank))x 50 μg/ml x (10mm/0.5 mm) X DF

Where, DF=20
Note
The DNA concentration is around 500 ug/mL but can be much lower (since the small volume of DNA primary aliquot is hard to be mixed), use the measured DNA value to calculate the DNA primary concentration.

Check absorbance of SG-II WS:
In a transparent microplate, load
(1) 200 uL Tris buffer as blank
(2) 10 uL SG-II WS and 190 uL Tris buffer
Read absorbance at 480 nm, the value after subtracted by blank shall be no higher than 0.21
Day 3: Read fluorescence
Day 3: Read fluorescence
Remove samples out of the fridge and allow to reach TemperatureRoom temperature for Duration00:02:00 before loading the microplate.

Note
Since fluorescence decreases with increasing temperature, with percentage changes depending on the fluorophore (Bashford, 1987), the SG-II WS must be kept dark at RT (22ºC) and the samples must be equilibrated at RT (c. 2 min).

Adhere black film on the top of a microplate lid.
Equipment
Black Vinyl Films for Fluorescence and Photoprotection
NAME
VWR
BRAND
89087-692
SKU

Equipment
Microplate Lids
NAME
Polystyrene
TYPE
Greiner Bio-One
BRAND
07000288
SKU

Load Amount10 µL SG-II WS to each well in the microplate with 0.5 mL tip of stepper, and cover the plate with the black-film lid.
Equipment
Finntip™ Stepper Pipette Tips
NAME
500 uL
TYPE
Thermo Scientific™
BRAND
9404170
SKU

Equipment
96-Well Black Microplates
NAME
Polystyrene
TYPE
Greiner Bio-One
BRAND
655076
SKU

Note
Wipe or dab the liquid drop on the outside of the tip, avoid wiping the tip open before dispensing the liquid.

Organize tubes in 96-well microtube rack in the same order as how microplates are loaded.

Load Amount190 µL working sample to the microplate by reverse pipetting.


Note
Wipe or dab the liquid drop on the outside of the tip, avoid wiping the tip open before dispensing the liquid.

45m
Shake black film covered microplate at TemperatureRoom temperature for Duration01:30:00
Note
Read fluorescence right after 1h30 incubation at room temperature.

1h 30m
Setup microplate reader:
Plate: Greiner F bottom chimney well PP 96 well;
Shake: Continuous 5s at 600 rpm
Bandwidth: 5 nm
Endpoint reading: Ex 490 nm/Em 520 nm;
Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU

Read fluorescence and export data to excel sheet.
In the fume hood, dispose any waste with SG-II into fluorescence stain waste container (some stain waste has DMSO solvent).
Calculate
Calculate
RNA standard curve
Concentrations of RNA standards in the microplate
RNA standardSecondary 2 ug/mL (uL)Tris (uL)STEB (uL)WS (uL)Sample in microplate (uL)SG-II (uL)Conc in microplate (ng/mL)
R10.00650.00250.0050.00190.0010.000.00
R210.00640.00250.0050.00190.0010.0020.00
R350.00600.00250.0050.00190.0010.00100.00
R4100.00550.00250.0050.00190.0010.00200.00
R5150.00500.00250.0050.00190.0010.00300.00

Slope of fluorescence in Set A vs concentration of RNA standard gives mRNA+DNase (≈0.03)
Slope of fluorescence in Set B vs concentration of RNA standard gives mRNA+RNase
Calculate ρ

Total RNA of the samples

Where,
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.
DNA standard curve
Concentrations of DNA standards in the microplate: Use measured DNA primary concentration instead of 500 ug/mL:

DNA primary Conc (ug/mL)DNA primary (uL)Tris (uL)Conc. DNA secondary (ug/mL)
595

DNA secondary Conc. (ug/mL)DNA secondary (uL)Tris (uL)Conc. DNA tertiary (ug/mL)
40960

DNA standardDNA tertiary (uL)Tris (uL)STEB (uL)WS (uL)Sample in microplate (uL)SG-II (uL)Conc. in microplate (ng/mL)
R1065025050190100
D1106402505019010~10
D2406102505019010~40
D31005502505019010~100
Slope of fluorescence in Set A vs concentration of DNA standard gives mDNA+DNase
Slope of fluorescence in Set B vs concentration of DNA standard gives mDNA+RNase (≈0.12)
Calculate δ

Total DNA of the samples

Where,
RFUB and RFUC are the fluorescence in Tube B and Tube C of the same sample
RFUBBlank and RFUCBlank are the fluorescence in Tube B and Tube C of the blank.
Dilution factor=40
If,
  • Sample is extracted by 1 mL extraction reagent
  • In Set 1, sample is diluted to 1/10
  • In Set 3, diluted by Tris and all working solutions to 250/950
  • In microplate, diluted by SG-II WS to 190/200

Citations
Berdalet E, Roldán C, Olivar MP, Lysnes K. Quantifying RNA and DNA in planktonic organisms with SYBR Green II and nucleases. Part A. Optimisation of the assay
https://doi.org/10.3989/scimar.2005.69n11
Berdalet E, Roldán C, Olivar MP. Quantifying RNA and DNA in planktonic organisms with SYBR Green II and nucleases. Part B. Quantification in natural samples
https://doi.org/10.3989/scimar.2005.69n117