Total RNA and DNA from Microalgae (24 samples per day)

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.

Equipment

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

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

Clean working surface with decontamination solution.

Prepare Tris buffer 5mM 8.0

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

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 )

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

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

Cap the package and invert for a thorough mix.

Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Store 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)

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

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

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 )

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

Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Store 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)

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

Use reverse pipetting to precisely aliquot 60 uL into 5 mL RNase free tube. Store at -20°C.

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

Clean working surface with decontamination solution.

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

Prepare STEB (1% )

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

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

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

Mix the following ingredients to obtain STEB (1% ):

sarcosine (20% ): 500µL

EDTA (0.5M ): 10µL

Tris buffer (5mM ,8.0): 9mL+490µL

Prepare ice bath

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

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

Equipment

Value	Label
Lysing tube matrix D	NAME
2 mL	TYPE
MP biomedical	BRAND
116913500	SKU
http://www.mpbio.com	LINK

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

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

Prior to placing sample filter into the bead tube:

Invert the tube then put back On ice.

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

Value	Label
CENTRIFUGE 5430 R	NAME
Eppendorf	BRAND
MP2231000510	SKU

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

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

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

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

Disrupt samples on the bead mill at 6.5 m/s

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

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

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

Centrifuge extracted samples 10000x g,4°C

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 10000x g,4°C

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 100µL 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.

Prepare ice bath.

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

Clean working surface with decontamination solution.

Prepare falcon tubes, microtubes and tube racks in biosafety cabinet| A | B | C | | --- | --- | --- | | 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

Value	Label
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 (5mM ,8.0) to the reagent tubes:

A	B
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

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

Prepare STEB (0.05% )

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

Add 250µL 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 -20On ice.

Important technique for accurately preparing standards and working solutions

Prepare DNA secondary standard solution 25ug/ml

Add 190µL 5 mM Tris buffer into DNA primary tube.

Prepare DNA tertiary standard solution 1ug/ml

Mix DNA secondary standard solution by aspiring up and down several times with pipet.

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

Keep 37On ice.

Prepare RNA secondary standard solution 25ug/ml

Add 210µL Tris buffer into RNA primary tube.

Prepare RNA tertiary standard solution 2ug/ml

Mix RNA secondary standard solution by aspiring up and down several times with pipet.

Transfer 120µL RNA secondary solution (25ug/ml) to RNA tertiary standard tube and mix.

Keep 37On 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

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

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 to . 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 to . Pay more attention to the solution mixing of primary RNA solution.

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

Equipment

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

Prepare 900mM MgCl₂

Pour 1M MgCl₂solution into 5 mL RNase free Falcon tube

Transfer 360µL 1M MgCl₂solution into 900 mM MgCl₂tube

Add 60µL 900mM MgCl₂ to WS-B

Prepare 900mM CaCl₂

Pour 1M CaCl₂solution into 5 mL RNase free Falcon tube

Transfer 360µL 1M CaCl₂solution into 900 mM CaCl₂tube

Add 60µL 900mM CaCl₂ to WS-B

Prepare SG-II WS

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

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)

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

Prepare RNase working solution 0.5mg/ml

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

Thoroughly mix RNase tube and then transfer 60µL 0.5mg/ml RNase working solution to WS-B.

Keep WS-B 37On ice .

Thoroughly mix RNase tube and then transfer 60µL 0.5mg/ml RNase working solution to WS-C.

Keep WS-C 37On ice .

Label DNase tube with "WS-A", add 2X1000+820 mL 5 mM Tris buffer into the tube.

Add 60µL 900mM MgCl₂ to WS-A

Add 60µL 900mM CaCl₂ to WS-A.

Keep WS-A 37On ice.

Use reverse pipetting: load 50µL WS-A to tubes in Set A .

Use reverse pipetting: load 50µL WS-A to tubes Set C .

Use reverse pipetting: load 50µL WS-B to tubes in Set B .

Use reverse pipetting: load 50µL WS-C to tubes in Set C .

Organize sample extracts (i.e., the 100 uL aliquot) into Set 1 .

Forward pipetting, add 900 uL 5mM 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.

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 0h 20m 0s.

After incubation, Invert each tube for thorough mixing, then place them into the freezer for 2 min to stop the reaction.

Remove samples out of the freezer and allow to reach Room temperature before loading the microplate.

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

Load 10µL 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

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

Reverse pipetting: load 190µL working samples to the microplate.

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

Setup microplate reader:Plate: Greiner F bottom chimney well PP 96 well;Shake: Continuous 5s at 600 rpmFluorescence bandwidth: 12 nm Exciation: 490 nmEmission: 520 nm Equipment

Value	Label
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).

RNA standard curve

Concentrations of RNA standards in the microplate: Use measured RNA primary concentration instead of 200 ug/mL:

A	B	C	D
	30	210

A	B	C	D
	120	1380

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

Slope of fluorescence in Set A vs concentration of RNA standard gives m _{RNA+DNase (~0.024)}

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

Calculate ρ (<=0.15)

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.

DNA standard curve

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

A	B	C	D
	10	190

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

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 (~0.13)

Calculate δ (<=0.15)

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.

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