Mar 24, 2022

Public workspaceTotal RNA and DNA from Microalgae (24 samples per day) V.10

DOI
dx.doi.org/10.17504/protocols.io.6qpvro85bvmk/v12
  • 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/v12
Protocol Citation: Ying-Yu Hu, Zoe V. Finkel 2022. Total RNA and DNA from Microalgae (24 samples per day) . protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvro85bvmk/v12Version 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: March 24, 2022
Last Modified: March 24, 2022
Protocol Integer ID: 59822
Keywords: RNA, DNA, SYBR Green II, DNase, RNase, microalgae, fluorescence
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 was set at ca. 40 ~300 ng RNA or 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
ReagentNuclease decontamination solutionIDTCatalog #11-05-03-01
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
ReagentDEOXYRIBONUCLEASE1 RNase and Protease FreeBioshopCatalog # DRB002.10
ReagentMagnesium chloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #63069-100ML
ReagentCalcium chloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #21115-100ML
ReagentN-Lauroylsarosine sodium salt solution (20% RNase/DNase free)Merck MilliporeSigma (Sigma-Aldrich)Catalog #L744-50mL
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 thymusMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4522-1MG
ReagentEDTA buffer (0.5M DNase/RNase free)BioshopCatalog #EDT333.100
ReagentNuclease decontamination solutionIDTCatalog #11-05-03-01
ReagentRibonuclease A from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #R6513-50MG
ReagentUltraPure™ DNase/RNase-Free Distilled WaterThermofisherCatalog #10977023
ReagentE. coli Total RNAThermo Fisher ScientificCatalog #AM7940
ReagentNuclease decontamination solutionIDTCatalog #11-05-03-01
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 and clean working surface with decontamination solution.
ReagentNuclease decontamination solutionVWR InternationalCatalog #11-05-03-01

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 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

Equipment
Microcentrifuge Tubes
NAME
1.7 mL/0.6 mL
TYPE
Axygen Scientific
BRAND
MCT-175-C/MCT-060-L-C
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

Dilute 5 ul primary DNA standard solution with 95 ul Tris buffer (Concentration5 mM ,Ph8.0 ) in a microtube (600 ul).
Measure DNA 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

Note
The dilution is to avoid saturated observation at 260 nm.

DNA concentration (μg/ml) = (Abs260-Abs260 (blank))x 50 μg/ml x (10mm/0.5 mm) X DF
Where, DF=20.
Note
If the measured DNA concentration is not close to Concentration500 ug/ml , check reverse pipetting technique.


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 100 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at Temperature-20 °C .

Equipment
Finntip stepper pipette tips
NAME
1250 ul (sterile)
TYPE
Thermo Scientific
BRAND
Thermo Scientific™ 9404183
SKU

Day 2: Exact RNA and DNA
Day 2: Exact RNA and DNA
Turn on UV light in biosafety cabinet for Duration00:15:00 and clean working surface with decontamination solution.
Prepare falcon tubes and tube rack in biosafety cabinet


AB
Volume of tube (mL)Contents in the tube
50.5 M EDTA
520% sarcosine
15 or 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 15 or 50 mL falcon tube.

Note
For large volume of Tris buffer usage, use sterile serological pipet


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.
Vortex immediately then put back TemperatureOn ice .
Equipment
VWR ANALOG VORTEX MIXER
NAME
VWR
BRAND
10153-838
SKU
With tube insert
SPECIFICATIONS

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 tubesTemperatureOn 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 tubesTemperatureOn 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 tubesTemperatureOn 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 150 uL of homogenate into RNase free 600 uL tube.


Freeze both aliquote (150 uL) and the rest of homogenate (in bead mill tube) 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 and clean working surface with decontamination solution.
Prepare falcon tubes, microtubes and tube racks in biosafety cabinet
* Maximum number of samples (including blanks) per assay is 20.

ABC
Number of tubesType of tubesContents
35 mL falcon tubes1 M MgCl2, 1 M CaCl2, Sybr Green II working solution (SG-II WS)
150 mL falcon tube5 mM Tris buffer
415 mL falcon tubes0.05% STEB, Working solution A (WS-A), Working solution B (WS-B), Working solution (WS-C)
51.7 mL RNase free tubesRNase working solution, Secondary RNA standard solution, Secondary DNA standard solution, 900mM MgCl2, 900 mM CaCl2
331.7 mL RNase free tubesRNA standard solutions for RNA standard curves, DNA standard soutions for DNA standard curves
N= total number of samples and blanks1.7 mL RNase free tubesSamples and blanks
3XN1.7 mL RNase free tubesDiluted samples and blanks
5Microtube racksTubes of 1.7 mL
1Tube racksFalcon tubes

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

Day 3: Run the assay
Day 3: Run the assay
Organize and label the tubes as shown below

Set 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)


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

AB
Reagent type5 mM Tris (uL)
0.05% STEB9X1000 + 500
RNA990+495
DNA998
RNase380
900 mM MgCl240
900 mM CaCl240
WS-A5X1000 + 640
WS-B5X1000 +640
WS-C5X1000 + 880
SG-II WS2X1000 + 500


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 2. The unit of volume is uL.

Follow the sheet, add Tris buffer (Concentration5 mM ,Ph8.0 ) to each tube in Set 3. 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 each tube in Set 2 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 Amount120 µL Concentration900 mM MgCl2 to WS-A and 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 Amount120 µL Concentration900 mM CaCl2 to WS-A and WS-B

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 Amount120 µL Concentration0.5 mg/ml RNase to WS-B and WS-C.
Keep WS-B and WS-C TemperatureOn ice .
Add Amount120 µL DNase primary stock solution ( Concentration5 mg/ml ) to WS-A.
Keep WS-A TemperatureOn ice .
Note
Two microtubes of aliquot (70 ul/tube)

Centrifuge extracted samples Centrifigation10000 x g, 4°C, 00:04:00
Take one tube of ReagentSYBR™ Green II RNA Gel Stain, 10,000X concentrate in DMSOVWR InternationalCatalog #S7564
out of the freezer, keep it at TemperatureRoom temperature .
If open a new package, wrap 1.7 mL microtube with foil and aliquot 1000 ul to each tube, store at Temperature-20 °C .

Prepare RNA secondary standard solution Concentration2 ug/ml
Add Amount15 µL RNA primary standard solution to RNA standard tube and vortex.
Keep TemperatureOn ice .
Prepare DNA secondary standard solution Concentration1 ug/ml
Add Amount2 µL DNA primary standard solution to DNA standard tube and vortex.
Keep TemperatureOn ice .
Load Amount50 µL WS-A to Tubes A in Set 2 and Set 3.
Note
From Step 51 to 54: Reverse pipetting
Decontaminate pipet between different WS.

Load Amount50 µL WS-A to Tubes C in Set 2 and Set 3.
Load Amount50 µL WS-B to Tubes B in Set 2 and Set 3.
Load Amount50 µL WS-C to Tubes C in Set 2 and Set 3.
Add Amount100 µL centrifuged samples to its corresponding tubes in Set 1.
Vortex each tube.
From Set 1, transfer Amount250 µL of diluted samples to each corresponding tubes in Set 3.
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.

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.


Vortex each tube for Duration00:00:02 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
Read fluorescence
Read fluorescence
Prepare SYBR Green II working solution (SG-II WS)
Each 96-well microplate requires 1 mL of SG-II WS.
Note
For 24 samples:
2.5 mL Tris
17.5 uL SG-II WS

Centrifuge one tube of SG-II concentrate at TemperatureRoom temperature Centrifigation13000 rpm, 00:05:00 to deposit DMSO.

5m
Wrap SG-II WS tube with foil, transfer Concentration17.5 ul supernatant of SYBR Green II 10,000X concentrate to SG-II WS tube in biosafety cabinet.
Note
Any step involving SYBR Green II should be operated in dark room or at least dim light.


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.

After incubation, vortex each tube for Duration00:00:02 and then place into the fridge to stop the reaction.
Allow samples 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).

Label the microplate.
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.
Blank must be included in each plate.
Pink area: RNA standard solutions for RNA standard curves
Blue area: DNA standard solutions for DNA standard curves
Yellow area: Samples and blanks



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 Duration00:10:00
Note
(1) The fluorescence of the RNA-bound SG-II is highly stable during the 0 - 180 min period
(2) The fluorescence of the DNA-bound SG-II varies during the first 10 min and remains stable during the 10-60 min period, but tended to decrease afterwards.
(3) In summary, readings are performed within the 10-60 min period following the SG-II addition;
meanwhile the samples are kept dark at RT (22ºC).

10m
Setup microplate reader:
Plate: Greiner F bottom chimney well PP 96 well;
Shake: Continuous 5s at 600 rpm
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.
Calculate
Calculate
RNA standard curve
Concentrations of RNA standards in the microplate

Slope of fluorescence in Tube A vs concentration of RNA standard gives mRNA+DNase (≈0.03)
Slope of fluorescence in Tube 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

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 (≈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