TITLE: Total RNA and DNA from Microalgae (12 samples per microplate) 
AUTHORS: Ying-Yu Hu

[DESCRIPTION]
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.

 

 








[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.
 
 
  

[STEPS]
SECTION: Sample collection
7. Freeze-dry samples. Store at -80 °C.
 

 

SECTION: Primary solutions
8. Turn on UV light in biosafety cabinet for 15 min 
SECTION: Primary solutions
9. Clean working surface with decontamination solution.
SECTION: Primary solutions
10. Prepare Tris buffer  5 mM pH 8.0
SECTION: Primary solutions
10.1. Pour 1 M pH 8.0 Tris into an RNase free 15 mL Falcon tube
 
 

SECTION: Primary solutions
10.2. Directly add 2.5 mL  1 MpH 8.0  Tris into 500 mL RNase free water in its original package.
 
 

SECTION: Primary solutions
11. RNA primary standard solution (200 ug/ml ) 
SECTION: Primary solutions
11.1. In the original package, the frozen E. Coli Total RNA is of 1 mg/mL, in which total RNA is 200 ug. 
 

 

SECTION: Primary solutions
11.2. Uncap the original package of E. Coli Total RNA and directly add 800 µL Tris buffer (5 mM ,pH 8.0) .
Cap the package and invert for a thorough mix.
 

SECTION: Primary solutions
11.3. Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Store at -80 °C
 

 

SECTION: Primary solutions
12. DNA primary standard solution (≈ 500 ug/ml)
SECTION: Primary solutions
12.1. Uncap the original package of Deoxyribonucleic acid from calf thymus and add   2 mL Tris buffer (5 mM ,pH 8.0).
 

 

SECTION: Primary solutions
12.2. Cap the package. Do not vortex or sonicate. 
SECTION: Primary solutions
12.3. Keep the solution in the fridge overnight to completely solubilize the DNA. Gentle reversion is recommended.
SECTION: Primary solutions
12.4. Aliquot 10 uL by stepper with sterile tip to 600 uL RNase free microtubes. Store at -20 °C
 

SECTION: Primary solutions
13. RNase primary stock solution (10 mg/ml )
SECTION: Primary solutions
13.1. Uncap the original package of Ribonuclease A from bovin pancreas and add  5 mL Tris buffer (5 mM ,pH 8.0).
 Cap the package and invert for a thorough mix.
 
SECTION: Primary solutions
13.2. Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Store at -20 °C
 

 

SECTION: Primary solutions
14. DNase primary stock solution (5 mg/ml = 10,000 U/mL)
SECTION: Primary solutions
14.1. Uncap the original package of Deoxyribonuclease1 and add  1 mL Tris buffer (5 mM ,pH 8.0) .
Cap the package and invert for a thorough mix.
 

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

 

SECTION: Total RNA and DNA extraction
15. Turn on UV light in biosafety cabinet for 15 min
SECTION: Total RNA and DNA extraction
16. Clean working surface with decontamination solution.
SECTION: Total RNA and DNA extraction
17. Prepare falcon tubes and tube rack in biosafety cabinet
 

 

 

SECTION: Total RNA and DNA extraction
18. Prepare STEB (1 % )
 

SECTION: Total RNA and DNA extraction
18.1. Pour sarcosine (20 % ) into an RNase free 5 mL falcon tube.
 
SECTION: Total RNA and DNA extraction
18.2. Pour EDTA (0.5 M ) into an RNase free 5 mL falcon tube.
 
SECTION: Total RNA and DNA extraction
18.3. Pour Tris buffer (5 mM ,pH 8.0)  into an RNase free 50 mL falcon tube.
SECTION: Total RNA and DNA extraction
18.4. Mix the following ingredients to obtain STEB (1 % ):
sarcosine (20 % ):  500 µL  
EDTA (0.5 M ):  10 µL 
Tris buffer (5 mM ,pH 8.0):  9 mL+490 µL  
SECTION: Total RNA and DNA extraction
19. Prepare ice bath
SECTION: Total RNA and DNA extraction
20. Remove freeze-dried samples from -80ºC freezer and place them on ice.
SECTION: Total RNA and DNA extraction
21. Add 500 µL Tris buffer (5 mM ,pH 8.0) and 500 µL STEB (1 % ) to the bead tube. Place tubes on ice.
 

 

SECTION: Total RNA and DNA extraction
22. Rinse forceps by  70 % volume ethanol and air dry.
 

SECTION: Total RNA and DNA extraction
23. Transfer sample/blank filter into the bead tube by using clean forceps.

Prior to placing sample filter into the bead tube: 
 

SECTION: Total RNA and DNA extraction
24. Invert the tube then put back on ice.
SECTION: Total RNA and DNA extraction
26. Disrupt samples on the bead mill at 6.5 m/s.
 

SECTION: Total RNA and DNA extraction
27. Keep tubes on ice. Check the label on each tube, restore the label if it fades.
SECTION: Total RNA and DNA extraction
28. Disrupt samples on the bead mill at 6.5 m/s.
SECTION: Total RNA and DNA extraction
29. Keep tubes on ice. Check the label on each tube, restore the label if it fades.
SECTION: Total RNA and DNA extraction
30. Disrupt samples on the bead mill at 6.5 m/s 
SECTION: Total RNA and DNA extraction
31. Keep tubes on ice. Check the label on each tube, restore the label if it fades.
SECTION: Total RNA and DNA extraction
32. Disrupt samples on the bead mill at 6.5 m/s.
SECTION: Total RNA and DNA extraction
33. Continuously shake homogenate in a multi-head vortex at the highest speed for 60 min Room temperature
 

SECTION: Total RNA and DNA extraction
39. In the biosafety cabinet, transfer supernatant for the assay.

SECTION: Total RNA and DNA extraction
40. Store both boxes of samples at -80 °C.
SECTION: Assay (Be prepared: it is a full working day procedure)
41. Prepare ice bath.
SECTION: Assay (Be prepared: it is a full working day procedure)
42. Turn on UV light in biosafety cabinet for 15 min 
SECTION: Assay (Be prepared: it is a full working day procedure)
43. Clean working surface with decontamination solution.
SECTION: Assay (Be prepared: it is a full working day procedure)
44. Prepare falcon tubes, microtubes and tube racks in biosafety cabinet
 

 

SECTION: Assay (Be prepared: it is a full working day procedure)
45. 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

 

 
SECTION: Assay (Be prepared: it is a full working day procedure)
46. Label tubes for reagents as following. 
Follow the sheet, add Tris buffer (5 mM ,pH 8.0) to the reagent tubes:

 

SECTION: Assay (Be prepared: it is a full working day procedure)
47. Thaw Sybr green II at room temperature
  
SECTION: Assay (Be prepared: it is a full working day procedure)
48. Add Tris buffer (5 mM ,pH 8.0) to each tube in Set A, B and C. The unit of volume is uL.

 

 

SECTION: Assay (Be prepared: it is a full working day procedure)
49. Prepare STEB (0.05 % ) 
Add 500 µL STEB (1 % ) to 0.05% STEB tube, and invert to mix.
SECTION: Assay (Be prepared: it is a full working day procedure)
50. Add 250 µL STEB (0.05 % ) to RNA and DNA standards in Set A, B and C  by reverse pipetting.

 

SECTION: Assay (Be prepared: it is a full working day procedure)
51. Place sample extract, RNase and DNase primary stock solutions, RNA and DNA primary standard solutions on ice.
SECTION: Assay (Be prepared: it is a full working day procedure)
63. Turn on shaker/incubator and set temperature to 37 °C.
 

SECTION: Assay (Be prepared: it is a full working day procedure)
64. Prepare 900 mM MgCl2
SECTION: Assay (Be prepared: it is a full working day procedure)
64.1. Pour 1 M MgCl2 solution into 5 mL RNase free Falcon tube
 

SECTION: Assay (Be prepared: it is a full working day procedure)
64.2. Transfer 360 µL 1 M MgCl2 solution into 900 mM MgCl2 tube
SECTION: Assay (Be prepared: it is a full working day procedure)
65. Add 60 µL  900 mM MgCl2 to WS-B 
SECTION: Assay (Be prepared: it is a full working day procedure)
66. Prepare  900 mM  CaCl2
SECTION: Assay (Be prepared: it is a full working day procedure)
66.1. Pour 1 M CaCl2 solution into 5 mL RNase free Falcon tube
 

SECTION: Assay (Be prepared: it is a full working day procedure)
66.2. Transfer 360 µL 1 M CaCl2 solution into 900 mM CaCl2 tube
SECTION: Assay (Be prepared: it is a full working day procedure)
67. Add 60 µL  900 mM  CaCl2 to WS-B 
SECTION: Assay (Be prepared: it is a full working day procedure)
68. Prepare SG-II WS
SECTION: Assay (Be prepared: it is a full working day procedure)
68.1. Centrifuge one tube of SG-II concentrate at Room temperature  13000 rpm, 5 min to deposit DMSO.
SECTION: Assay (Be prepared: it is a full working day procedure)
68.2. 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)
 

SECTION: Assay (Be prepared: it is a full working day procedure)
70. 
 

SECTION: Assay (Be prepared: it is a full working day procedure)
71. Prepare RNase working solution 0.5 mg/ml 
Add 20 µL RNase primary stock solution (10 mg/ml) to RNase tube 

SECTION: Assay (Be prepared: it is a full working day procedure)
72. Thoroughly mix RNase tube and then transfer 60 µL  0.5 mg/ml  RNase working solution to WS-B.
Keep WS-B on ice .
SECTION: Assay (Be prepared: it is a full working day procedure)
73. Thoroughly mix RNase tube and then transfer 60 µL  0.5 mg/ml  RNase working solution to WS-C.
Keep WS-C on ice .
SECTION: Assay (Be prepared: it is a full working day procedure)
76. Add 60 µL  900 mM  CaCl2 to WS-A.
Keep WS-A on ice.
SECTION: Assay (Be prepared: it is a full working day procedure)
53. Prepare DNA secondary standard solution 25 ug/ml
SECTION: Assay (Be prepared: it is a full working day procedure)
77. Use reverse pipetting: load 50 µL WS-A to tubes in Set A.

SECTION: Assay (Be prepared: it is a full working day procedure)
78. Use reverse pipetting: load 50 µL WS-A to tubes Set C.
SECTION: Assay (Be prepared: it is a full working day procedure)
79. Use reverse pipetting: load 50 µL WS-B to tubes in Set B.
SECTION: Assay (Be prepared: it is a full working day procedure)
80. Use reverse pipetting: load 50 µL WS-C to tubes in Set C.
SECTION: Assay (Be prepared: it is a full working day procedure)
81. Organize sample extracts (i.e., the 100 uL aliquot) into Set 1. 
Forward pipetting, add 900 uL 5 mM Tris buffer into each tube.

 

SECTION: Assay (Be prepared: it is a full working day procedure)
82. 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.

 


 

SECTION: Assay (Be prepared: it is a full working day procedure)
83. 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.

 

SECTION: Assay (Be prepared: it is a full working day procedure)
85. After incubation, Invert each tube for thorough mixing, then place them into the freezer for 2 min to stop the reaction.
SECTION: Assay (Be prepared: it is a full working day procedure)
69. Check absorbance of SG-II WS:
SECTION: Assay (Be prepared: it is a full working day procedure)
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
SECTION: Assay (Be prepared: it is a full working day procedure)
69.2. Read absorbance at 480 nm, the value after subtracted by blank shall be no higher than 0.21
SECTION: Fluorescence measurement
86. Remove samples out of the freezer and allow to reach Room temperature before loading the microplate.

 

SECTION: Fluorescence measurement
87. Adhere black film on the top of a microplate lid.
 

 

SECTION: Fluorescence measurement
88. 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.
 

SECTION: Fluorescence measurement
89. Reverse pipetting: load 190 µL working samples to the microplate.
 
 

 
 

SECTION: Fluorescence measurement
90.  Shake black film covered microplate at 37 Room temperature for 90 min

 

SECTION: Fluorescence measurement
91. 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
 

 

SECTION: Fluorescence measurement
92. Read fluorescence and export data to excel sheet.
SECTION: Fluorescence measurement
93. In the fume hood, dispose any waste with SG-II into fluorescence stain waste container (some stain waste has DMSO solvent).
SECTION: Calculation
94. RNA standard curve
SECTION: Calculation
94.1. Concentrations of RNA standards in the microplate: Use measured RNA primary concentration instead of 200 ug/mL:

 

 
 
 
 
 
SECTION: Calculation
94.2. 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
SECTION: Calculation
94.3. Calculate ρ (<=0.15)
 
SECTION: Calculation
95. 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.
SECTION: Calculation
96. DNA standard curve 
SECTION: Calculation
96.1. 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)1595

 2nd (uL)Tris (uL)STEBWS (uL)Conc (ng/mL)Sample (uL)SG II (uL)Final (ng/mL)R10650250500.00190100D1106402505011.311901010.74D2406102505045.221901042.96D310055025050113.0519010107.4
  
SECTION: Calculation
96.2. 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)
SECTION: Calculation
96.3. Calculate δ (<=0.15)
 
SECTION: Calculation
97. 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.
SECTION: Calculation
98. 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

SECTION: Assay (Be prepared: it is a full working day procedure)
53.1. Add 190 µL 5 mM Tris buffer into DNA primary tube.
SECTION: Assay (Be prepared: it is a full working day procedure)
61.  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.
SECTION: Assay (Be prepared: it is a full working day procedure)
54. Prepare DNA tertiary standard solution 1 ug/ml 
SECTION: Assay (Be prepared: it is a full working day procedure)
58. Before loading the samples, use pipet tip to aspire and dispense multiple times for thorough mix. 
SECTION: Assay (Be prepared: it is a full working day procedure)
60. Read absorbance at 260 and 320 nm.
SECTION: Assay (Be prepared: it is a full working day procedure)
84. Place all tubes into the shaker/incubator at 37 °C, continuously shaking at 200 RPM for 20 min.
 

SECTION: Assay (Be prepared: it is a full working day procedure)
55. Prepare RNA secondary standard solution 25 ug/ml
SECTION: Assay (Be prepared: it is a full working day procedure)
55.1. Add 210 µL Tris buffer into RNA primary tube. 

SECTION: Assay (Be prepared: it is a full working day procedure)
56. Prepare RNA tertiary standard solution 2 ug/ml 

SECTION: Assay (Be prepared: it is a full working day procedure)
57. Remove the DNA and RNA secondary out of the biosafety cabinet.
SECTION: Assay (Be prepared: it is a full working day procedure)
62.  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.
SECTION: Total RNA and DNA extraction
25. Turn on refrigerated centrifuge and set the temperature to 4 °C.
 

SECTION: Total RNA and DNA extraction
34. Centrifuge extracted samples  10000 x g, 4 min, 4 °C 
SECTION: Total RNA and DNA extraction
39.1. Invert the tube and mix thoroughly, then aspire 100 µL of supernatant using the reverse pipetting technique.
SECTION: Total RNA and DNA extraction
39.2. Dispense this 100 uL into the 2 mL microtube.
SECTION: Total RNA and DNA extraction
39.3. Return any remaining extract in the tip back to the extraction tube.
SECTION: Total RNA and DNA extraction
35. In the biosafety cabinet, prepare 3XN 2 mL RNase free microtubes, N = number of samples.
SECTION: Assay (Be prepared: it is a full working day procedure)
52. Important technique for accurately preparing standards and working solutions

 

SECTION: Assay (Be prepared: it is a full working day procedure)
59. Use reverse pipetting:
Load 4 ul DNA and RNA secondary onto the μdrop plate, in duplicate.
 

 

SECTION: Total RNA and DNA extraction
36. In the biosafety cabinet, transfer all extract to their corresponding microtubes.
SECTION: Total RNA and DNA extraction
37. Centrifuge extracted samples  10000 x g, 4 min, 4 °C 
SECTION: Total RNA and DNA extraction
38. In the biosafety cabinet, transfer supernatant as much as possible to their corresponding microtubes, avoid disturbing the debris.
SECTION: Assay (Be prepared: it is a full working day procedure)
54.1. Mix DNA secondary standard solution by aspiring up and down several times with pipet.
 

SECTION: Assay (Be prepared: it is a full working day procedure)
54.2. Transfer 40 µL DNA secondary solution (25 ug/ml) to DNA tertiary standard tube. 
Keep on ice.
SECTION: Assay (Be prepared: it is a full working day procedure)
56.1. Mix RNA secondary standard solution by aspiring up and down several times with pipet.
 

SECTION: Assay (Be prepared: it is a full working day procedure)
56.2. Transfer 120 µL RNA secondary solution (25 ug/ml) to RNA tertiary standard tube and mix. 
Keep on ice.
SECTION: Assay (Be prepared: it is a full working day procedure)
74. Label DNase tube with "WS-A", add 2X1000+820 mL 5 mM Tris buffer into the tube.
SECTION: Assay (Be prepared: it is a full working day procedure)
75. Add 60 µL  900 mM MgCl2 to WS-A
SECTION: Sample collection
1. Filter microalgae in liquid media onto polycarbonate filters, using gentle vacuum pressure (130 mmHg).
SECTION: Sample collection
2. Rinse filter tunnel with filtered artificial seawater (nutrient free) to avoid sample loss.
SECTION: Sample collection
3. 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.
SECTION: Sample collection
4. Place folded filter in 2 mL cryogenic vial.
SECTION: Sample collection
5. Blank is not required in this measurement.
SECTION: Sample collection
6. Flash-freeze tubes with liquid nitrogen and store at -80 °C 
SECTION: Total RNA and DNA extraction
39.4. Place the aliquot and the remaining extract into two separate boxes, i.e., one for aliquot only, the other one for remaining extract.
SECTION: Assay (Be prepared: it is a full working day procedure)
82.1. Load diluted samples to each corresponding tubes (marked in yellow) in Set A, B and C.

SECTION: Assay (Be prepared: it is a full working day procedure)
82.2. Add RNA tertiary standard to tubes (marked in pink) in Set A, B and C.
SECTION: Assay (Be prepared: it is a full working day procedure)
82.3. Add DNA tertiary standard to tubes (marked in blue) in Set A, B and C.