Total RNA and DNA from Microalgae (12 samples per microplate)

Ying-Yu Hu, Zoe V. Finkel

Published: 2023-02-20 DOI: 10.17504/protocols.io.6qpvro85bvmk/v11

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 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 https://doi.org/10.3989/scimar.2005.69n117

Before start

Steps

Day 1: Freeze-dry samples

Freeze dry samples and blank filters. Freeze at -80°C until processed.

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

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

Day 1: Prepare primary solutions

Turn on UV light in biosafety cabinet for 0h 15m 0s

Clean working surface with decontamination solution.

Prepare Tris buffer 5mM 8.0

4.1.

Pour 1M 8.0 Tris into an RNase free 15 mL Falcon tube

Equipment

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

4.2.

Directly add 2.5mL 1M``8.0 Tris into 500 mL RNase free water in its original package.

Equipment

Value	Label
BT Barrier Pipet Tips	NAME
Pre-Sterile	TYPE
Neptune®	BRAND
BT1250, BT100, BT10	SKU

RNA primary standard solution (200ug/ml )

5.1.

In the original package, the frozen E. Coli Total RNA is of 1 mg/mL, in which total RNA is 200 ug.

Note

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/sp_7940.pdf

5.2.

Uncap the original package of E. Coli Total RNA and directly add 800µL Tris buffer (5mM ,8.0) .

Cap the package and vortex for a thorough mix.

5.3.

Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at -80°C

Equipment

Value	Label
Finnpipette Stepper Pipette	NAME
Thermo Scientific™	BRAND
4540000	SKU
https://www.fishersci.com/us/en/home.html	LINK

Equipment

Value	Label
Finntip stepper pipette tips	NAME
500 ul (sterile)	TYPE
Thermo Scientific	BRAND
Thermo Scientific™ 9404173	SKU
https://www.fishersci.com/us/en/home.html	LINK

DNA primary standard solution (≈ 500ug/ml)

6.1.

Uncap the original package of Deoxyribonucleic acid from calf thymus and add 2mL Tris buffer (5mM ,8.0).

Note

https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/d4522pis.pdf

6.2.

Cap the package. Do not vortex or sonicate.

6.3.

Keep the solution at 0°C ~4°C overnight to completely solubilize the DNA. Gentle reversion is recommended.

6.4.

Aliquot 10 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at -80°C

Equipment

Value	Label
Finntip stepper pipette tips	NAME
500 ul (sterile)	TYPE
Thermo Scientific	BRAND
Thermo Scientific™ 9404173	SKU
https://www.fishersci.com/us/en/home.html	LINK

RNase primary stock solution (10mg/ml )

7.1.

Uncap the original package of Ribonuclease A from bovin pancreas and add 5mL Tris buffer (5mM ,8.0). Cap the package and vortex for a thorough mix.

7.2.

Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at -20°C

Equipment

Value	Label
Finntip stepper pipette tips	NAME
500 ul (sterile)	TYPE
Thermo Scientific	BRAND
Thermo Scientific™ 9404173	SKU
https://www.fishersci.com/us/en/home.html	LINK

Equipment

Value	Label
Finntip™ Stepper Pipette Tips	NAME
500 ul (Sterile)	TYPE
Thermo Scientific	BRAND
21-377-149	SKU
https://www.fishersci.com/us/en/home.html	LINK

DNase primary stock solution (5mg/ml = 10,000 U/mL)

8.1.

Uncap the original package of Deoxyribonuclease1 and add 1mL Tris buffer (5mM ,8.0) .Cap the package and vortex for a thorough mix.

8.2.

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

Day 2: Exact RNA and DNA

Turn on UV light in biosafety cabinet for 0h 15m 0s

10.

Clean working surface with decontamination solution.

11.

Prepare falcon tubes and tube rack in biosafety cabinet| A | B | | --- | --- | | 5 | 0.5 M EDTA | | 5 | 20% sarcosine | | 50 | 5 mM Tris | | 15 or 50 | 1% STEB |

Equipment

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

Equipment

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

12.

Prepare STEB (1% )

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

12.1.

Pour sarcosine (20% ) into an RNase free 5 mL falcon tube.

12.2.

Pour EDTA (0.5M ) into an RNase free 5 mL falcon tube.

12.3.

Pour Tris buffer (5mM ,8.0) into an RNase free 50 mL falcon tube.

12.4.

Mix 500µL sarcosine (20% ) , 10µL EDTA (0.5M ) and 9mL+490µL Tris buffer (5mM ,8.0) to obtain STEB (1% ).

13.

Prepare ice bath

14.

Remove freeze-dried samples from -80ºC freezer and place them On ice.

15.

Add 500µL Tris buffer (5mM ,8.0) and 500µL STEB (1% ) to the bead tube. Place tubes On ice.

Equipment

Value	Label
LYSING TUBES	NAME
MATRIX D 2 mL/15 mL	TYPE
MP BIOMEDICALS	BRAND
116913500/116933050	SKU

16.

Rinse forceps by 70% volume ethanol and air dry.

Equipment

Value	Label
Filter forceps	NAME
blunt end, stainless steel	TYPE
Millipore	BRAND
XX6200006P	SKU
http://www.emdmillipore.com/	LINK

17.

Transfer sample/blank filter into the bead tube by using clean forceps.

18.

Invert immediately then put back On ice.

19.

Disrupt samples on the bead mill at 6.5 m/s. Equipment

Value	Label
Fastprep-24 5G™ Sample Preparation Instrument	NAME
MP Biomedicals	BRAND
116005500	SKU

20.

Keep tubes On ice. Check the label on each tube, restore the label if it fades.

21.

Disrupt samples on the bead mill at 6.5 m/s.

22.

Keep tubes On ice. Check the label on each tube, restore the label if it fades.

23.

Disrupt samples on the bead mill at 6.5 m/s

24.

Keep tubes On ice. Check the label on each tube, restore the label if it fades.

25.

Disrupt samples on the bead mill at 6.5 m/s.

26.

Continuously shake homogenate in a multi-head vortex at the highest speed for 1h 0m 0s Room temperature

Note

Votex mixer should be able to remain stable on the bench under this vortex speed.

27.

In the biosafety cabinet, transfer all homogenate into RNase free 2 mL micro-tube.

28.

Freeze at -80°C until analyzed.

Day 3: Run the assay

29.

Prepare ice bath.

30.

Turn on UV light in biosafety cabinet for 0h 15m 0s

31.

Clean working surface with decontamination solution.

32.

Prepare falcon tubes, microtubes and tube racks in biosafety cabinet| A | B | C | | --- | --- | --- | | 5 | 5 mL falcon tubes | 1 M MgCl2 | | | | 1 M CaCl2 | | | | Working solution A (WS-A) | | | | 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 secondary standard solution | | | | DNA tertiary standard solution | | | | 900 mM MgCl2 | | | | 900 mM CaCl2 | | | | Sybr green working solution (SG-II WS) | | 1 | 600 uL RNase free tube | DNA secondary standard | | 24 | 2 mL RNase free tubes | RNA standard solutions for RNA standard curves | | | | DNA standard solutions for DNA standard curves | | N= total number of samples and blanks | 2 mL RNase free tubes | Samples and blanks | | 3XN | 2 mL RNase free tubes | Diluted samples and blanks | | 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

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

Day 3: Run the assay (Caution: It is a long procedure!)

33.

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

34.

Label tubes for reagents as following.

Follow the sheet, add Tris buffer (5mM ,8.0) to the reagent tubes:

A	B
SG-II WS	1000+250
WS-A	2X1000+820
WS-B	2X1000+820
WS-C	2X1000+940
RNase	380
900 mM MgCl2	40
900 mM CaCl2	40
RNA secondary	990+495
DNA secondary	95
DNA tertiary	960
0.05% STEB	9X1000 + 500

35.

Thaw Sybr green II at room temperature

36.

Add 900µL Tris buffer (5mM ,8.0) to each tube in Set 1

Note

Depending on the dilution of extracted sample

37.

Follow the sheet, add Tris buffer (5mM ,8.0) to each tube in Set A, B and C . The unit of volume is uL.

38.

Prepare STEB (0.05% )

Add 500µL STEB (1% ) to 0.05% STEB tube, and vortex.

39.

Add 250µL STEB (0.05% ) to RNA and DNA standards in Set A, B and C by reverse pipetting.

40.

Place RNase and DNase primary stock solutions, RNA and DNA primary standard solutions and samples -20On ice.

41.

Turn on refrigerated centrifuge and set the temperature to 4°C. Equipment

Value	Label
CENTRIFUGE 5430 R	NAME
Eppendorf	BRAND
MP2231000510	SKU

42.

Turn on shaker/incubator and set temperature to 37°C.

Equipment

Value	Label
SHAKING INCUBATOR	NAME
71L	TYPE
Corning® LSE™	BRAND
6753	SKU

43.

Prepare 900mM MgCl₂

43.1.

Pour 1M MgCl₂solution into 5 mL RNase free Falcon tube

43.2.

Transfer 360µL 1M MgCl₂solution into 900 mM MgCl₂tube

44.

Add 60µL 900mM MgCl₂ to WS-A

45.

Add 60µL 900mM MgCl₂ to WS-B

46.

Prepare 900mM CaCl₂

46.1.

Pour 1M CaCl₂solution into 5 mL RNase free Falcon tube

46.2.

Transfer 360µL 1M CaCl₂solution into 900 mM CaCl₂tube

47.

Add 60µL 900mM CaCl₂ to WS-A

48.

Add 60µL 900mM CaCl₂ to WS-B

49.

Prepare SG-II WS

49.1.

Centrifuge one tube of SG-II concentrate at Room temperature 13000rpm to deposit DMSO.

49.2.

Wrap SG-II WS tube with foil, transfer supernatant supernatant of SYBR Green II 10,000X concentrate to SG-II WS tube in biosafety cabinet ( 8.75ul 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.

50.

Note

Lunch break!

51.

Prepare RNase working solution 0.5mg/ml

Add 20µL RNase primary stock solution (10mg/ml) to RNase tube

52.

Add 60µL 0.5mg/ml RNase to WS-B.

Keep WS-B 37On ice .

53.

Add 60µL 0.5mg/ml RNase to WS-C.

Keep WS-C 37On ice .

54.

Add 60µL DNase primary stock solution ( 5mg/ml ) to WS-A.

Keep WS-A 37On ice.

55.

Centrifuge extracted samples 10000x g,4°C

56.

Prepare RNA secondary standard solution 2ug/ml

Add 15µL RNA primary standard solution to RNA standard tube and mix.

Keep 37On ice .

57.

Prepare DNA secondary standard solution (25ug/ml)

Add 5µL primary DNA standard solution (500ug/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.

58.

Prepare DNA tertiary standard solution 1ug/ml

Add 40µL DNA secondary solution (25ug/ml) to DNA tertiary standard tube and mix.

Keep 37On ice.

59.

Load 50µL WS-A to tubes in Set A .

Note

From 59 to 62: Reverse pipettingDecontaminate pipet between different WS.

60.

Load 50µL WS-A to tubes Set C .

61.

Load 50µL WS-B to tubes in Set B .

62.

Load 50µL WS-C to tubes in Set C .

63.

Add 100µL centrifuged samples to its corresponding tubes in Set 1 .

Gently invert the tube to mix sample.

64.

From Set 1, transfer 250µL 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.

65.

Invert each tube to mix well and place all tubes into the shaker/incubator at 37°C, continuously shaking at 200 RPM for 0h 20m 0s.

Note

Incubation time is critical. Temperature might be disturbed by door open/close. Don't start the timer until temperature returns to 37°C.

66.

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

67.

Measure DNA secondary concentration by using μdrop plate (sample volume: 4 ul)

Use Tris buffer (5mM ,8.0) as blank.

Equipment

Value	Label
µDrop™ Plates	NAME
Thermo Scientific	BRAND
N12391	SKU
https://www.lifetechnologies.com	LINK

Equipment

Value	Label
Varioskan LUX Multimode Microplate Reader	NAME
Thermo Fisher	BRAND
VL0L00D0	SKU

68.

DNA_primary concentration (μg/ml) = (Abs₂₆₀-Abs_{260 (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.

69.

Check absorbance of SG-II WS:

69.1.

In a transparent microplate, load

(1) 200 uL Tris buffer as blank

(2) 10 uL SG-II WS and 190 uL Tris buffer

69.2.

Read absorbance at 480 nm, the value after subtracted by blank shall be no higher than 0.21

Day 3: Read fluorescence

70.

Remove samples out of the fridge and allow to reach Room temperature for 0h 2m 0s 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).

71.

Adhere black film on the top of a microplate lid.

Equipment

Value	Label
Black Vinyl Films for Fluorescence and Photoprotection	NAME
VWR	BRAND
89087-692	SKU

Equipment

Value	Label
Microplate Lids	NAME
Polystyrene	TYPE
Greiner Bio-One	BRAND
07000288	SKU
https://www.fishersci.com/us/en/home.html	LINK

72.

Load 10µL 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

Value	Label
Finntip™ Stepper Pipette Tips	NAME
500 uL	TYPE
Thermo Scientific™	BRAND
9404170	SKU
https://www.fishersci.com/us/en/home.html	LINK

Equipment

Value	Label
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.

73.

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

74.

Load 190µL 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.

75.

Shake black film covered microplate at 37Room temperature for 1h 30m 0s

Note

Read fluorescence right after 1h30 incubation at room temperature.

76.

Setup microplate reader:Plate: Greiner F bottom chimney well PP 96 well;Shake: Continuous 5s at 600 rpmBandwidth: 5 nmEndpoint reading: Ex 490 nm/Em 520 nm; Equipment

Value	Label
Varioskan LUX Multimode Microplate Reader	NAME
Thermo Fisher	BRAND
VL0L00D0	SKU

77.

Read fluorescence and export data to excel sheet.

78.

In the fume hood, dispose any waste with SG-II into fluorescence stain waste container (some stain waste has DMSO solvent).

Calculate

79.

RNA standard curve

79.1.

Concentrations of RNA standards in the microplate

A	B	C	D	E	F	G	H
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

79.2.

Slope of fluorescence in Set A vs concentration of RNA standard gives m _RNA+DNase(≈0.03)

Slope of fluorescence in Set B vs concentration of RNA standard gives m_RNA+RNase

79.3.

Calculate ρ

80.

Total RNA of the samples

Where,

RFU_A and RFU_C are the fluorescence in Tube A and Tube C of the same sample.

RFU_ABlank and RFU_CBlank are the fluorescence in Tube A and Tube _C of the blank.

81.

DNA standard curve

81.1.

Concentrations of DNA standards in the microplate: Use measured DNA primary concentration instead of 500 ug/mL:

A	B	C	D
	5	95

A	B	C	D
	40	960

A	B	C	D	E	F	G	H
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

81.2.

Slope of fluorescence in Set A vs concentration of DNA standard gives m _DNA+DNase

Slope of fluorescence in Set B vs concentration of DNA standard gives m _DNA+RNase (≈0.12)

81.3.

Calculate δ

82.

Total DNA of the samples

Where,

RFU_B and RFU_C are the fluorescence in Tube B and Tube C of the same sample

RFU_BBlank and RFU_CBlank are the fluorescence in Tube B and Tube _C of the blank.

83.

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

Total RNA and DNA from Microalgae (12 samples per microplate)

Abstract

Before start

Steps

Day 1: Freeze-dry samples

Day 1: Prepare primary solutions

Day 2: Exact RNA and DNA

Day 3: Run the assay

Day 3: Run the assay (Caution: It is a long procedure!)

Day 3: Verify DNA concentration and SG-II absorbance

Day 3: Read fluorescence

Calculate

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