Feb 27, 2024
Version 12
Total RNA and DNA from Microalgae (12 samples per microplate) V.12
- 1Dalhousie University
Protocol Citation: Ying-Yu Hu 2024. Total RNA and DNA from Microalgae (12 samples per microplate) . 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 15, 2023
Last Modified: February 27, 2024
Protocol Integer ID: 78848
Keywords: RNA, DNA, SYBR Green II, DNase, RNase, microalgae, fluorescence
Funders Acknowledgement:
Simons Foundation
Grant ID: 549937
Simons Foundation
Grant ID: 723789
Abstract
Presented herein is a protocol for the extraction and quantification of bulk RNA and DNA from microalgae, adapted from the methodology outlined by Berdalet et al. (2005). RNA and DNA are extracted from microalgae samples and quantified using the fluorochrome SYBR Green II. To ensure accuracy, the concentrations of RNA and DNA standards are determined via absorbance measurements at 260 nm and 320 nm. This additional step aids in maintaining the consistency of the standard curve coefficients (i.e., the slope). The method demonstrates a sensitivity range of approximately 20-700 ng/ml for RNA and 10-700 ng/ml for DNA in the assay.
CITATION
CITATION
Guidelines
(1) Estimation of RNA/DNA in the collected microalgae samples:
L-LOD for RNA and DNA is 20 and 10 ng/mL, respectively. Common dilution from sample collected on the filter to assay is 1/40. The minimum RNA and DNA per filter requires to be no less than 1 and 0.5 ug/filter, respectively.
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).
(2) Verification of DNA and RNA standard concentrations ensures highly consistent assay results, facilitating comparisons of RNA and DNA data across researchers within the same group or across different groups. Below is an example showcasing the RNA and DNA values of Tripos obtained on different processing days, with sample replicates collected from various bottles. It's important to note that due to the large cell volume, cell counting of Tripos exhibits higher variation compared to small cells that can be counted using machines. This variation contributes to the standard deviation observed in RNA and DNA values.
| Date of assay | RNA (pg/cell) | SD of RNA | DNA (pg/cell) | SD of DNA | RNA:DNA | SD of the ratio | |
| 20240110 | 221.48 | 62.22 | 342.65 | 63.05 | 0.64 | 0.07 | |
| 20240108 | 219.90 | 50.71 | 355.13 | 58.78 | 0.61 | 0.05 | |
| 20240105 | 214.58 | 46.92 | 377.48 | 60.65 | 0.57 | 0.03 | |
| 20240104 | 197.81 | 61.88 | 324.47 | 73.52 | 0.60 | 0.08 |
Protocol materials
UltraPure™ DNase/RNase-Free Distilled WaterThermofisherCatalog #10977023
Step 10.2
SYBR™ Green II RNA Gel Stain, 10,000X concentrate in DMSOThermo FisherCatalog #S7564
Step 47
Calcium chloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #21115-100ML
Step 66.1
Ribonuclease A from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #R6513-50MG
Step 13.1
Tris(hydroxymethyl)aminomethane hydrochloride 1M pH 8.0 RNase freeFisher ScientificCatalog #AAJ60080AK
Step 10.1
E. coli Total RNAThermo Fisher ScientificCatalog #AM7940
Step 11.1
Deoxyribonucleic acid from calf thymusMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4522-1MG
Step 12.1
N-Lauroylsarosine sodium salt solution (20% RNase/DNase free)Merck MilliporeSigma (Sigma-Aldrich)Catalog #L744-50mL
Step 18.1
EDTA buffer (0.5M DNase/RNase free)BioshopCatalog #EDT333.100
Step 18.2
Magnesium chloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #63069-100ML
Step 64.1
DEOXYRIBONUCLEASE1 RNase and Protease FreeBioshopCatalog # DRB002.10
Step 14.1
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.
Sample collection
Sample collection
Filter microalgae in liquid media onto polycarbonate filters, using gentle vacuum pressure (130 mmHg).
Rinse filter tunnel with filtered artificial seawater (nutrient free) to avoid sample loss.
Fold the filter with two tweezers:
(1) Fold in half along its diameter, creating a semicircular shape;
(2) Fold once more in the same direction, resulting in a long strip;
(3) Fold once more, halving its length, so that sample is secured.
Place folded filter in 2 mL cryogenic vial.
Blank is not required in this measurement.
Flash-freeze tubes with liquid nitrogen and store at -80 °C
Freeze-dry samples. Store at -80 °C .
Note
- 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.
Equipment
FreeZone® 2.5 L Benchtop Freeze Dryers
NAME
Labconco®
BRAND
700202000
SKU
Primary solutions
Primary solutions
Turn on UV light in biosafety cabinet for 00:15:00
Clean working surface with decontamination solution.
Prepare Tris buffer 5 mM 8.0
Pour 1 M 8.0 Tris into an RNase free 15 mL Falcon tube
Tris(hydroxymethyl)aminomethane hydrochloride 1M pH 8.0 RNase freeFisher ScientificCatalog #AAJ60080AK
Equipment
Falcon® Centrifuge Tubes
NAME
Polypropylene, Sterile, 15 mL
TYPE
Corning®
BRAND
352096
SKU
Directly add 2.5 mL 1 M 8.0 Tris into 500 mL RNase free water in its original package.
UltraPure™ DNase/RNase-Free Distilled WaterThermofisherCatalog #10977023
Equipment
BT Barrier Pipet Tips
NAME
Pre-Sterile
TYPE
Neptune®
BRAND
BT1250, BT100, BT10
SKU
RNA primary standard solution (200 ug/ml )
In the original package, the frozen E. Coli Total RNA is of 1 mg/mL, in which total RNA is 200 ug.
E. coli Total RNAThermo Fisher ScientificCatalog #AM7940
Note
Uncap the original package of E. Coli Total RNA and directly add 800 µL Tris buffer (5 mM ,8.0 ) .
Cap the package and invert for a thorough mix.
Note
Be aware of the package. If there is a conical bottom vial insert, add 400 ul Tris buffer first, cap the package, invert for a thorough mix.
Transfer the solution to a 2 mL RNase free tube.
Add another 400 ul Tris buffer, cap the package, invert for a thorough mix.
Transfer and combine the solutions together in the 2 mL tube.
Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Store at -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 (≈ 500 ug/ml )
Uncap the original package of Deoxyribonucleic acid from calf thymus and add 2 mL Tris buffer (5 mM ,8.0 ).
Deoxyribonucleic acid from calf thymusMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4522-1MG
Note
Cap the package. Do not vortex or sonicate.
Keep the solution in the fridge overnight to completely solubilize the DNA. Gentle reversion is recommended.
Aliquot 10 uL by stepper with sterile tip to 600 uL RNase free microtubes. Store at -20 °C
Equipment
Finntip stepper pipette tips
NAME
500 ul (sterile)
TYPE
Thermo Scientific
BRAND
Thermo Scientific™ 9404173
SKU
RNase primary stock solution (10 mg/ml )
Uncap the original package of Ribonuclease A from bovin pancreas and add 5 mL Tris buffer (5 mM ,8.0 ).
Cap the package and invert for a thorough mix.
Ribonuclease A from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #R6513-50MG
Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Store at -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 (5 mg/ml = 10,000 U/mL)
Uncap the original package of Deoxyribonuclease1 and add 1 mL Tris buffer (5 mM ,8.0 ) .
Cap the package and invert for a thorough mix.
DEOXYRIBONUCLEASE1 RNase and Protease FreeBioshopCatalog # DRB002.10
Use reverse pipetting to precisely aliquot 60 uL into 5 mL RNase free tube. Store at -20 °C .
Note
The 5 mL tube will be used as WS-A in the assay directly.
One package of DNase can be used for 16 assays.
Total RNA and DNA extraction
Total RNA and DNA extraction
Turn on UV light in biosafety cabinet for 00:15:00
Clean working surface with decontamination solution.
Prepare falcon tubes and tube rack in biosafety cabinet
| Volume of the tube (mL) | Content in the tube | |
| 5 | 0.5 M EDTA | |
| 5 | 20% sarcosine | |
| 50 | 5 mM Tris | |
| 15 or 50 | 1% 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 (1 % )
Note
Use the following formula to determine the total volume of 1% STEB required:
# samples X (500 ul) + (500 ul) = total volume of 1% STEB required
Pour sarcosine (20 % ) into an RNase free 5 mL falcon tube.
N-Lauroylsarosine sodium salt solution (20% RNase/DNase free)Merck MilliporeSigma (Sigma-Aldrich)Catalog #L744-50mL
Pour EDTA (0.5 M ) into an RNase free 5 mL falcon tube.
EDTA buffer (0.5M DNase/RNase free)BioshopCatalog #EDT333.100
Pour Tris buffer (5 mM ,8.0 ) into an RNase free 50 mL falcon tube.
Mix the following ingredients to obtain STEB (1 % ):
sarcosine (20 % ): 500 µL
EDTA (0.5 M ): 10 µL
Tris buffer (5 mM ,8.0 ): 9 mL +490 µL
Prepare ice bath
Remove freeze-dried samples from -80ºC freezer and place them On ice .
Add 500 µL Tris buffer (5 mM ,8.0 ) and 500 µL STEB (1 % ) to the bead tube. Place tubes On ice .
Equipment
Lysing tube matrix D
NAME
2 mL
TYPE
MP biomedical
BRAND
116913500
SKU
Note
Use 2 mL bead tube for both 25 mm and 47 mm size filter.
Rinse forceps by 70 % 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.
Prior to placing sample filter into the bead tube:
Note
(1) For filters folded into half-strip, unfold once to return to a strip.
(2) For filters folded into quarter-circles, unfold once to return to a half-circle shape, then fold once along the dimension to form a strip.
(2) For filters haphazardly into a compact mass, carefully unfold with two tweezers (avoiding losing biomass), fold once into a half-cricle shape, then fold once more along the dimension to form a strip
Invert the tube then put back On ice .
20s
Turn on refrigerated centrifuge and set the temperature to 4 °C .
Equipment
CENTRIFUGE 5430 R
NAME
Eppendorf
BRAND
MP2231000510
SKU
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 On 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 On 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 On 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 01:00:00 Room temperature
Note
Votex mixer should be able to remain stable on the bench under this vortex speed.
1h
Centrifuge extracted samples 10000 x g, 4°C, 00:04:00
4m
In the biosafety cabinet, prepare 3XN 2 mL RNase free microtubes, N = number of samples.
In the biosafety cabinet, transfer all extract to their corresponding microtubes.
Centrifuge extracted samples 10000 x g, 4°C, 00:04:00
4m
In the biosafety cabinet, transfer supernatant as much as possible to their corresponding microtubes, avoid disturbing the debris.
In the biosafety cabinet, transfer supernatant for the assay.
Invert the tube and mix thoroughly, then aspire 100 µL of supernatant using the reverse pipetting technique.
Dispense this 100 uL into the 2 mL microtube.
Return any remaining extract in the tip back to the extraction tube.
Place the aliquot and the remaining extract into two separate boxes, i.e., one for aliquot only, the other one for remaining extract.
Store both boxes of samples at -80 °C .
Assay (Be prepared: it is a full working day procedure)
Assay (Be prepared: it is a full working day procedure)
Prepare ice bath.
Turn on UV light in biosafety cabinet for 00:15:00
Clean working surface with decontamination solution.
Prepare falcon tubes, microtubes and tube racks in biosafety cabinet
| Number of tubes | Type of tubes | Contents | |
| 4 | 5 mL falcon tubes | 1 M MgCl2 | |
| 1 M CaCl2 | |||
| Working solution B (WS-B) | |||
| Working solution C (WS-C) | |||
| 1 | 50 mL falcon tube | 5 mM Tris buffer | |
| 1 | 15 mL falcon tubes | 0.05% STEB | |
| 6 | 2 mL RNase free tubes | RNase working solution | |
| RNA tertiary standard solution | |||
| DNA tertiary standard solution | |||
| 900 mM MgCl2 | |||
| 900 mM CaCl2 | |||
| Sybr green working solution (SG-II WS) | |||
| 24 | 2 mL RNase free tubes | RNA standard solutions for RNA standard curves | |
| DNA standard solutions for DNA standard curves | |||
| 3XN (N = Number of samples) | 2 mL RNase free tubes | Diluted samples | |
| 4 | Microtube racks | Tubes 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 | |||
| 1 | Tube racks | Falcon tubes |
Equipment
Screw-Cap Centrifuge Tube
NAME
5 mL
TYPE
VWR
BRAND
10002-738
SKU
Organize and label the tubes as shown below, log sample numbers into the tube rack layout.
Set 1:
This tube rack holds sample extract (100 uL) 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 (5 mM ,8.0 ) to the reagent tubes:
| Content | 5 mM Tris (uL) | |
| SG-II WS | 1000+250 | |
| WS-B | 2X1000+820 | |
| WS-C | 2X1000+940 | |
| RNase | 380 | |
| 900 mM MgCl2 | 40 | |
| 900 mM CaCl2 | 40 | |
| RNA tertiary | 690X2 | |
| DNA tertiary | 960 | |
| 0.05% STEB | 9X1000 + 500 |
Thaw Sybr green II at room temperature
SYBR™ Green II RNA Gel Stain, 10,000X concentrate in DMSOThermo FisherCatalog #S7564
Add Tris buffer (5 mM ,8.0 ) to each tube in Set A, B and C. The unit of volume is uL.
Note
Avoid changing the dilution for samples in this step. The total volume required for samples in Set A, B and C is already 750 ul.
Prepare STEB (0.05 % )
Add 500 µL STEB (1 % ) to 0.05% STEB tube, and invert to mix.
Add 250 µL STEB (0.05 % ) to RNA and DNA standards in Set A, B and C by reverse pipetting.
Place sample extract, RNase and DNase primary stock solutions, RNA and DNA primary standard solutions On ice .
Important technique for accurately preparing standards and working solutions
Note
Be aware of the conical shaped microtubes. The conical shaped bottom often retains a small volume of liquid due to surface tension. This residual liquid can impact the accuracy of measurements or the concentration of the solution.
(1) For inverting mix:
Gently invert the tube several times to ensure that any residual liquid at the bottom is mixed back into the solution.
(2) For pipet mix:
Place the pipette tip all the way to the bottom of the conical tube and then aspire/dispense multiple times.
Prepare DNA secondary standard solution 25 ug/ml
Add 190 µL 5 mM Tris buffer into DNA primary tube.
Prepare DNA tertiary standard solution 1 ug/ml
Mix DNA secondary standard solution by aspiring up and down several times with pipet.
Note
Do not vortex!
Transfer 40 µL DNA secondary solution (25 ug/ml ) to DNA tertiary standard tube.
Keep On ice .
Prepare RNA secondary standard solution 25 ug/ml
Add 210 µL Tris buffer into RNA primary tube.
Prepare RNA tertiary standard solution 2 ug/ml
Mix RNA secondary standard solution by aspiring up and down several times with pipet.
Note
Do not vortex!
Transfer 120 µL RNA secondary solution (25 ug/ml ) to RNA tertiary standard tube and mix.
Keep On ice .
Remove the DNA and RNA secondary out of the biosafety cabinet.
Before loading the samples, use pipet tip to aspire and dispense multiple times for thorough mix.
Use reverse pipetting:
Load 4 ul DNA and RNA secondary onto the μdrop plate, in duplicate.
Equipment
µDrop™ Plates
NAME
Thermo Scientific
BRAND
N12391
SKU
Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU
Read absorbance at 260 and 320 nm.
DNA_primary concentration (μg/ml) = (Abs260-Abs320)x 50 μg/ml x (10mm/0.49 mm) X DF
Where, DF=20.
The DNA primary concentration should be around 500 ug/mL.
If the DNA primary concentration is less than 400 (reading from udrop is less than 0.055) or higher than 600 ug/mL, repeat go to step #53 to go to step #60 . Pay more attention to the solution mixing of primary DNA solution.
RNA_primary concentration (μg/ml) = (Abs260-Abs320)x 40 μg/ml x (10mm/0.49 mm) X DF
Where, DF=8.
The DNA primary concentration should be around 200 ug/mL.
If the DNA primary concentration is less than 150 (reading from udrop is less than 0.055) or higher than 250 ug/mL, repeat go to step #56 to go to step #60 . Pay more attention to the solution mixing of primary RNA solution.
Turn on shaker/incubator and set temperature to 37 °C .
Equipment
SHAKING INCUBATOR
NAME
71L
TYPE
Corning® LSE™
BRAND
6753
SKU
Prepare 900 mM MgCl2
Pour 1 M MgCl2 solution into 5 mL RNase free Falcon tube
Magnesium chloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #63069-100ML
Transfer 360 µL 1 M MgCl2 solution into 900 mM MgCl2 tube
Add 60 µL 900 mM MgCl2 to WS-B
Prepare 900 mM CaCl2
Pour 1 M CaCl2 solution into 5 mL RNase free Falcon tube
Calcium chloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #21115-100ML
Transfer 360 µL 1 M CaCl2 solution into 900 mM CaCl2 tube
Add 60 µL 900 mM CaCl2 to WS-B
Prepare SG-II WS
Centrifuge one tube of SG-II concentrate at Room temperature 13000 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 ( 8.75 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.
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
Note
Lunch break!
Prepare RNase working solution 0.5 mg/ml
Add 20 µL RNase primary stock solution (10 mg/ml ) to RNase tube
Thoroughly mix RNase tube and then transfer 60 µL 0.5 mg/ml RNase working solution to WS-B.
Keep WS-B On ice .
Thoroughly mix RNase tube and then transfer 60 µL 0.5 mg/ml RNase working solution to WS-C.
Keep WS-C On ice .
Label DNase tube with "WS-A", add 2X1000+820 mL 5 mM Tris buffer into the tube.
Add 60 µL 900 mM MgCl2 to WS-A
Add 60 µL 900 mM CaCl2 to WS-A.
Keep WS-A On ice .
Use reverse pipetting: load 50 µL WS-A to tubes in Set A.
Use reverse pipetting: load 50 µL WS-A to tubes Set C.
Use reverse pipetting: load 50 µL WS-B to tubes in Set B.
Use reverse pipetting: load 50 µL WS-C to tubes in Set C.
Organize sample extracts (i.e., the 100 uL aliquot) into Set 1.
Forward pipetting, add 900 uL 5 mM Tris buffer into each tube.
Thoroughly mix sample prior to transferring.
Follow the layout below to add diluted samples, RNA tertiary and DNA tertiary into Set A, B and C. The unit of volume is uL.
Note
Forward pipetting:
(1) To avoid enzyme cross-contamination:
Renew tip between sets when dispensing the same sample or standard tertiary solution
(2) Aspire up and down for a complete dispense and thorough mix
Load diluted samples to each corresponding tubes (marked in yellow) in Set A, B and C.
Add RNA tertiary standard to tubes (marked in pink) in Set A, B and C.
Add DNA tertiary standard to tubes (marked in blue) in Set A, B and C.
Invert each tube for thorough mixing, then organize the tubes in a 96-well microtube rack following the same order as the microplates are loaded.
Place all tubes into the shaker/incubator at 37 °C , continuously shaking at 200 RPM for 00:20:00 .
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 thorough mixing, then place them into the freezer for 2 min to stop the reaction.
Fluorescence measurement
Fluorescence measurement
Remove samples out of the freezer and allow to reach Room temperature 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 10 µL SG-II WS to each well by either 0.5 mL stepper or 10 uL pipet. Cover the plate with the black-film lid.
Equipment
96-Well Black Microplates
NAME
Polystyrene
TYPE
Greiner Bio-One
BRAND
655076
SKU
Reverse pipetting: load 190 µL working samples to the microplate.
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 Room temperature for 01:30:00
Note
The fluorescence value and the ratio between RNA and DNA stabilize within 90 minutes, contrary to the 10 minutes reported in the original paper.
1h 30m
Setup microplate reader:
Plate: Greiner F bottom chimney well PP 96 well;
Shake: Continuous 5s at 600 rpm
Fluorescence bandwidth: 12 nm
Exciation: 490 nm
Emission: 520 nm
Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU
Note
Bandwidth at 12 nm gives more consistent results compared to 5 nm bandwidth.
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).
Calculation
Calculation
RNA standard curve
Concentrations of RNA standards in the microplate: Use measured RNA primary concentration instead of 200 ug/mL:
| RNA primary conc. (ug/mL) | RNA primary (uL) | Tris (uL) | Conc. RNA secondary (ug/mL) | |
| 30 | 210 |
| RNA secondary conc. (ug/mL) | RNA secondary (uL) | Tris (uL) | Conc. RNA tertiary (ug/mL) | |
| 120 | 1380 |
| RNA standard | Tertiary (uL) | Tris (uL) | STEB (uL) | WS (uL) | Sample in microplate (uL) | SG-II (uL) | Conc in microplate (ng/mL) | |
| R1 | 0.00 | 650.00 | 250.00 | 50.00 | 190.00 | 10.00 | 0.00 | |
| R2 | 10.00 | 640.00 | 250.00 | 50.00 | 190.00 | 10.00 | ~20.00 | |
| R3 | 50.00 | 600.00 | 250.00 | 50.00 | 190.00 | 10.00 | ~100.00 | |
| R4 | 100.00 | 550.00 | 250.00 | 50.00 | 190.00 | 10.00 | ~200.00 | |
| R5 | 150.00 | 500.00 | 250.00 | 50.00 | 190.00 | 10.00 | ~300.00 |
Slope of fluorescence in Set A vs concentration of RNA standard gives mRNA+DNase (~0.024)
Slope of fluorescence in Set B vs concentration of RNA standard gives mRNA+RNase
Calculate ρ (<=0.15)
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) | |
| 10 | 190 |
| DNA secondary Conc. (ug/mL) | DNA secondary (uL) | Tris (uL) | Conc. DNA tertiary (ug/mL) | |
| 40 | 960 |
| DNA standard | DNA tertiary (uL) | Tris (uL) | STEB (uL) | WS (uL) | Sample in microplate (uL) | SG-II (uL) | Conc. in microplate (ng/mL) | |
| R1 | 0 | 650 | 250 | 50 | 190 | 10 | 0 | |
| D1 | 10 | 640 | 250 | 50 | 190 | 10 | ~10 | |
| D2 | 40 | 610 | 250 | 50 | 190 | 10 | ~40 | |
| D3 | 100 | 550 | 250 | 50 | 190 | 10 | ~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 (~0.13)
Calculate δ (<=0.15)
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 100/1000
- In Set 3, diluted by Tris and all working solutions to 250/950
- In microplate, diluted by SG-II WS to 190/200