TITLE: Total RNA and DNA from Microalgae (12 samples per microplate) 
AUTHORS: Ying-Yu Hu, Zoe V. Finkel

[DESCRIPTION]
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.

 
  
  
 

 








[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.

[STEPS]
SECTION: Day 1: Freeze-dry samples
1. Freeze dry samples and blank filters. Freeze at -80 °C  until processed.
 

 

SECTION: Day 1: Prepare primary solutions
2. Turn on UV light in biosafety cabinet for 15 min 
SECTION: Day 1: Prepare primary solutions
4. Prepare Tris buffer  5 mM pH 8.0
SECTION: Day 1: Prepare primary solutions
4.1. Pour 1 M pH 8.0 Tris into an RNase free 15 mL Falcon tube
 
 

SECTION: Day 1: Prepare primary solutions
4.2. Directly add 2.5 mL  1 MpH 8.0  Tris into 500 mL RNase free water in its original package.
 
 

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

 

SECTION: Day 1: Prepare primary solutions
5.2. 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 vortex for a thorough mix.
SECTION: Day 1: Prepare primary solutions
5.3. Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at -80 °C
 

 

SECTION: Day 1: Prepare primary solutions
6. DNA primary standard solution (≈ 500 ug/ml)
SECTION: Day 1: Prepare primary solutions
6.1. Uncap the original package of Deoxyribonucleic acid from calf thymus and add   2 mL Tris buffer (5 mM ,pH 8.0).
 

 

SECTION: Day 1: Prepare primary solutions
6.2. Cap the package. Do not vortex or sonicate. 
SECTION: Day 1: Prepare primary solutions
6.3. Keep the solution at 0 °C ~4 °C overnight to completely solubilize the DNA. Gentle reversion is recommended.
SECTION: Day 1: Prepare primary solutions
6.4. Aliquot 10 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at -80 °C
 

SECTION: Day 1: Prepare primary solutions
7. RNase primary stock solution (10 mg/ml )
SECTION: Day 1: Prepare primary solutions
7.1. 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 vortex for a thorough mix.
 
SECTION: Day 1: Prepare primary solutions
7.2. Aliquot 30 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at -20 °C
 

 

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

SECTION: Day 1: Prepare primary solutions
8.2. Aliquot 70 uL to 600 uL RNase free microtubes (every assay required 60 uL). Keep frozen at -20 °C.

SECTION: Day 2: Exact RNA and DNA
9. Turn on UV light in biosafety cabinet for 15 min
SECTION: Day 2: Exact RNA and DNA
11. Prepare falcon tubes and tube rack in biosafety cabinet
 

 

 

SECTION: Day 2: Exact RNA and DNA
12. Prepare STEB (1 % )
 

SECTION: Day 2: Exact RNA and DNA
12.1. Pour sarcosine (20 % ) into an RNase free 5 mL falcon tube.
 
SECTION: Day 2: Exact RNA and DNA
12.2. Pour EDTA (0.5 M ) into an RNase free 5 mL falcon tube.
 
SECTION: Day 2: Exact RNA and DNA
12.3. Pour Tris buffer (5 mM ,pH 8.0)  into an RNase free 50 mL falcon tube.
SECTION: Day 2: Exact RNA and DNA
12.4. Mix 500 µL sarcosine (20 % ) , 10 µL EDTA (0.5 M )  and 9 mL+490 µL Tris buffer (5 mM ,pH 8.0)  to obtain STEB (1 % ).
SECTION: Day 2: Exact RNA and DNA
13. Prepare ice bath
SECTION: Day 2: Exact RNA and DNA
14. Remove freeze-dried samples from -80ºC freezer and place them on ice.
SECTION: Day 2: Exact RNA and DNA
15. Add 500 µL Tris buffer (5 mM ,pH 8.0) and 500 µL STEB (1 % ) to the bead tube. Place tubes on ice.
 

SECTION: Day 2: Exact RNA and DNA
16. Rinse forceps by  70 % volume ethanol and air dry.
 

SECTION: Day 2: Exact RNA and DNA
17. Transfer sample/blank filter into the bead tube by using clean forceps.
SECTION: Day 2: Exact RNA and DNA
18. Invert immediately then put back on ice.
SECTION: Day 2: Exact RNA and DNA
19. Disrupt samples on the bead mill at 6.5 m/s.
 

SECTION: Day 2: Exact RNA and DNA
20. Keep tubes on ice. Check the label on each tube, restore the label if it fades.
SECTION: Day 2: Exact RNA and DNA
21. Disrupt samples on the bead mill at 6.5 m/s.
SECTION: Day 2: Exact RNA and DNA
22. Keep tubes on ice. Check the label on each tube, restore the label if it fades.
SECTION: Day 2: Exact RNA and DNA
23. Disrupt samples on the bead mill at 6.5 m/s 
SECTION: Day 2: Exact RNA and DNA
24. Keep tubes on ice. Check the label on each tube, restore the label if it fades.
SECTION: Day 2: Exact RNA and DNA
25. Disrupt samples on the bead mill at 6.5 m/s.
SECTION: Day 2: Exact RNA and DNA
26. Continuously shake homogenate in a multi-head vortex at the highest speed for 60 min Room temperature
 

SECTION: Day 2: Exact RNA and DNA
27. In the biosafety cabinet, transfer all homogenate into RNase free 2 mL micro-tube. 
SECTION: Day 2: Exact RNA and DNA
28. Freeze at -80 °C until analyzed.
SECTION: Day 3: Run the assay
29. Prepare ice bath.
SECTION: Day 3: Run the assay
30. Turn on UV light in biosafety cabinet for 15 min 
32. Prepare falcon tubes, microtubes and tube racks in biosafety cabinet
 

 

SECTION: 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

    
    

SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
34. Label tubes for reagents as following. 
Follow the sheet, add Tris buffer (5 mM ,pH 8.0) to the reagent tubes:

 


SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
36. Add 900 µL Tris buffer (5 mM ,pH 8.0) to each tube in Set 1

 

SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
37. Follow the sheet, add Tris buffer (5 mM ,pH 8.0) to each tube in Set A, B and C. The unit of volume is uL.

        
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
38. Prepare STEB (0.05 % ) 
Add 500 µL STEB (1 % ) to 0.05% STEB tube, and vortex.
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
39. Add 250 µL STEB (0.05 % ) to RNA and DNA standards in Set A, B and C  by reverse pipetting.
  
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
40. Place RNase and DNase primary stock solutions, RNA and DNA primary standard solutions and samples on ice.
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
41. Turn on refrigerated centrifuge and set the temperature to 4 °C.
 

SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
42. Turn on shaker/incubator and set temperature to 37 °C.
 

SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
43. Prepare 900 mM MgCl2
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
43.1. Pour 1 M MgCl2 solution into 5 mL RNase free Falcon tube
 

SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
43.2. Transfer 360 µL 1 M MgCl2 solution into 900 mM MgCl2 tube
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
44. Add 60 µL  900 mM MgCl2 to WS-A
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
46. Prepare  900 mM  CaCl2
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
46.1. Pour 1 M CaCl2 solution into 5 mL RNase free Falcon tube
 

SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
46.2. Transfer 360 µL 1 M CaCl2 solution into 900 mM CaCl2 tube
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
47. Add 60 µL  900 mM  CaCl2 to WS-A 
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
50. 
 

SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
51. Prepare RNase working solution 0.5 mg/ml 
Add 20 µL RNase primary stock solution (10 mg/ml) to RNase tube 

SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
52. Add 60 µL  0.5 mg/ml  RNase to WS-B.
Keep WS-B on ice .
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
54. Add 60 µL DNase primary stock solution ( 5 mg/ml ) to WS-A.
Keep WS-A on ice.
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
55. Centrifuge extracted samples  10000 x g, 4 min, 4 °C 
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
56. Prepare RNA secondary standard solution 2 ug/ml 
Add 15 µL RNA primary standard solution to RNA standard tube and mix. 
Keep on ice .
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
58. Prepare DNA tertiary standard solution 1 ug/ml 
Add 40 µL DNA secondary solution (25 ug/ml) to DNA tertiary standard tube and mix. 
Keep on ice.

SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
59. Load 50 µL WS-A to tubes in Set A.
 

SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
60. Load 50 µL WS-A to tubes Set C.
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
61. Load 50 µL WS-B to tubes in Set B.
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
62. Load 50 µL WS-C to tubes in Set C.
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
63. Add 100 µL centrifuged samples to its corresponding tubes in Set 1. 
Gently invert the tube to mix sample.
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
64. From Set 1, transfer 250 µL 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.
  
 


SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
65. Invert each tube to mix well and place all tubes into the shaker/incubator at 37 °C, continuously shaking at 200 RPM for 20 min.
 

SECTION: Day 3: Read fluorescence
71. Adhere black film on the top of a microplate lid.
 

 

SECTION: Day 3: Read fluorescence
70. Remove samples out of the fridge and allow to reach Room temperature for 2 min before loading the microplate.

 

SECTION: Day 3: Read fluorescence
73. Organize tubes in 96-well microtube rack in the same order as how microplates are loaded.
  
SECTION: Day 3: Read fluorescence
74. Load 190 µL working sample to the microplate by reverse pipetting.

 
   
 
 

SECTION: Day 3: Read fluorescence
75.  Shake black film covered microplate at 37 Room temperature for 90 min
 
  
SECTION: Day 3: Read fluorescence
76. Setup microplate reader:
Plate: Greiner F bottom chimney well PP 96 well;
Shake: Continuous 5s at 600 rpm
Bandwidth: 5 nm
Endpoint reading: Ex 490 nm/Em 520 nm;
 

SECTION: Day 3: Read fluorescence
77. Read fluorescence and export data to excel sheet.
SECTION: Calculate
79. RNA standard curve
SECTION: Calculate
79.1. Concentrations of RNA standards in the microplate
 
2 ug/mL (uL)Tris (uL)STEB (uL)WS (uL)Conc (ng/mL)sampleSG IIFinal (ng/mL)R10.00650.00250.0050.000.00190.0010.000.00R210.00640.00250.0050.0021.05190.0010.0020.00R350.00600.00250.0050.00105.26190.0010.00100.00R4100.00550.00250.0050.00210.53190.0010.00200.00R5150.00500.00250.0050.00315.79 190.0010.00300.00
 

SECTION: Calculate
79.2. Slope of fluorescence in Set A vs concentration of RNA standard gives mRNA+DNase (≈0.03)
Slope of fluorescence in Set B vs concentration of RNA standard gives mRNA+RNase
SECTION: Calculate
79.3. Calculate ρ
  
SECTION: Calculate
80. 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: Calculate
81. DNA standard curve 
SECTION: Calculate
81.1. 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: Calculate
81.2. 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 (≈0.12) 
SECTION: Calculate
81.3. Calculate δ
  
SECTION: Calculate
82. 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: Calculate
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

SECTION: 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 (5 mM ,pH 8.0) as blank.
 

 

SECTION: Day 3: Verify DNA concentration and SG-II absorbance
68. DNA_primary concentration (μg/ml) = (Abs260-Abs260 (blank))x 50 μg/ml x (10mm/0.5 mm) X DF

Where, DF=20
 

SECTION: Day 3: Verify DNA concentration and SG-II absorbance
69. Check absorbance of SG-II WS:
SECTION: Day 3: Verify DNA concentration and SG-II absorbance
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: Day 3: Verify DNA concentration and SG-II absorbance
69.2. Read absorbance at 480 nm, the value after subtracted by blank shall be no higher than 0.21
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
35. Thaw Sybr green II at room temperature
  
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
49. Prepare SG-II WS
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
49.1. Centrifuge one tube of SG-II concentrate at Room temperature  13000 rpm, 5 min to deposit DMSO.
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
49.2. 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: Day 3: Read fluorescence
72. Load 10 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.
 

 

 

SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
45. Add 60 µL  900 mM MgCl2 to WS-B 
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
48. Add 60 µL  900 mM  CaCl2 to WS-B 
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
53. Add 60 µL  0.5 mg/ml  RNase to WS-C.
Keep WS-C on ice .
SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
57. Prepare DNA secondary standard solution (25 ug/ml)
Add 5 µL primary DNA standard solution (500 ug/ml) to DNA secondary tube and mix. 

 

 

SECTION: Day 3: Run the assay (Caution: It is a long procedure!)
66. After incubation, invert each tube for mixing and then place into the fridge to stop the reaction.
SECTION: Day 3: Run the assay
31. Clean working surface with decontamination solution.
SECTION: Day 3: Read fluorescence
78. In the fume hood, dispose any waste with SG-II into fluorescence stain waste container (some stain waste has DMSO solvent).
SECTION: Day 2: Exact RNA and DNA
10. Clean working surface with decontamination solution.
SECTION: Day 1: Prepare primary solutions
3. Clean working surface with decontamination solution.