TITLE: Total RNA and DNA in Microalgae
AUTHORS: Yingyu  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. 
 

 





[GUIDELINES]
The optimized linear range in this assay for DNA is 0~100 ng/mL and RNA is 0~300 ng/mL in the microplate, respectively. Samples are diluted to 1/40 in the assay, therefore, maximum DNA and RNA required in the assay is around 4 ug and 12 ug per sample. Assuming total DNA and RNA is 1% and 5.7% of total biomass at replete condition, carbon per sample required in this assay is about 80 ug.

Microalgae samples are collected on polycarbonate filter and kept frozen under -80 ºC.


[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 and clean working surface with decontamination solution.
 

SECTION: Day 1: Prepare primary solutions
3. Prepare Tris buffer  5 mM pH 8.0
SECTION: Day 1: Prepare primary solutions
3.1. Pour 1 M pH 8.0 Tris into an RNase free 15 mL Falcon tube
 

 


SECTION: Day 1: Prepare primary solutions
3.2. 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
4. RNA primary standard solution (200 ug/ml ) 
SECTION: Day 1: Prepare primary solutions
4.1. In the original package, the E. Coli Total RNA is of 1 mg/mL, in which total RNA is 200 ug. 
 

 

SECTION: Day 1: Prepare primary solutions
4.2. 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
4.3. 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
5. DNA primary standard solution (≈ 500 ug/ml)
SECTION: Day 1: Prepare primary solutions
5.1. 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
5.2. Cap the package. Do not vortex or sonicate. 
SECTION: Day 1: Prepare primary solutions
5.3. Keep the solution at 0 °C ~4 °C overnight to completely solubilize the DNA. Gentle reversion is recommended.
SECTION: Day 1: Prepare primary solutions
5.4. 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
5.5. Dilute 5 ul primary DNA standard solution with 95 ul Tris buffer (5 mM ,pH 8.0) in a microtube (600 ul).
Measure DNA concentration by using μdrop plate (sample volume: 4 ul)
Use Tris buffer (5 mM ,pH 8.0) as blank.

 

 

 

SECTION: Day 1: Prepare primary solutions
5.6. DNA concentration (μg/ml) = (Abs260-Abs260 (blank))x 50 μg/ml x (10mm/0.5 mm) X DF
Where, DF=20.
 

SECTION: Day 1: Prepare primary solutions
6. RNase primary stock solution (10 mg/ml )
SECTION: Day 1: Prepare primary solutions
6.1. 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
6.2. 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
7. DNase primary stock solution (5 mg/ml = 10,000 U/mL)
SECTION: Day 1: Prepare primary solutions
7.1. 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
7.2. Aliquot 100 uL by stepper with sterile tip to 600 uL RNase free microtubes. Keep frozen at -20 °C.

 

SECTION: Day 2: Exact RNA and DNA
8. Turn on UV light in biosafety cabinet for 15 min and clean working surface with decontamination solution.
SECTION: Day 2: Exact RNA and DNA
9. Prepare falcon tubes and tube rack in biosafety cabinet


 

 

 

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

SECTION: Day 2: Exact RNA and DNA
10.1. Pour sarcosine (20 % ) into an RNase free 5 mL falcon tube.
 

SECTION: Day 2: Exact RNA and DNA
10.2. Pour EDTA (0.5 M ) into an RNase free 5 mL falcon tube.
 

SECTION: Day 2: Exact RNA and DNA
10.3. Pour Tris buffer (5 mM ,pH 8.0)  into an RNase free 15 mL falcon tube.

SECTION: Day 2: Exact RNA and DNA
10.4. 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
11. Prepare ice bath
SECTION: Day 2: Exact RNA and DNA
12. Remove freeze-dried samples from -80ºC freezer and place them on ice.
SECTION: Day 2: Exact RNA and DNA
13. 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
14. Rinse forceps by  70 % volume ethanol and air dry.
 

SECTION: Day 2: Exact RNA and DNA
15. Transfer sample/blank filter into the bead tube by using clean forceps.
SECTION: Day 2: Exact RNA and DNA
16. Vortex immediately then put back on ice.
 

SECTION: Day 2: Exact RNA and DNA
17. Disrupt samples on the bead mill at 6.5 m/s.
 

SECTION: Day 2: Exact RNA and DNA
18. Keep tubeson ice. Check the label on each tube, restore the label if it fades.
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 tubeson 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 tubeson 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. Continuously shake homogenate in a multi-head vortex at the highest speed for 60 min Room temperature
 

SECTION: Day 2: Exact RNA and DNA
25. In the biosafety cabinet, aliquot 150 uL of homogenates into RNase free 600 uL tubes. 
 

SECTION: Day 2: Exact RNA and DNA
26. Freeze at -80 °C until analyzed.
SECTION: Day 3: Run the assay
27. Prepare ice bath.
SECTION: Day 3: Run the assay
28. Turn on UV light in biosafety cabinet for 15 min and clean working surface with decontamination solution.
SECTION: Day 3: Run the assay
29. Prepare falcon tubes, microtubes and tube racks in biosafety cabinet
* Maximum number of samples (including blanks) per assay is 20.

 

 
 

SECTION: Day 3: Run the assay
30. Organize and label the tubes as shown below

Set 1: 
In microtube rack, label 1.7 mL tubes for samples and blanks to be further diluted.

  
Set 2:
In microtube rack, label 1.7 mL tubes for RNA (marked in pink) and DNA (marked in blue) standard solutions to be used as standard curves.
Tubes A is for standard solutions treated with working solution A (WS-A)
Tubes B is for standard solutions treated with working solution B (WS-B)
Tubes C is for standard solutions treated with working solution C (WS-C)

     
Set 3:
In microtube rack, label 1.7 mL tubes for diluted samples and blanks.
Tubes A is for diluted samples and blanks treated with working solution A (WS-A)
Tubes B is for diluted samples and blanks treated with working solution B (WS-B)
Tubes C is for diluted samples and blanks treated with working solution C (WS-C)

   
SECTION: Day 3: Run the assay
31. 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
32. Add 900 µL Tris buffer (5 mM ,pH 8.0) to each tube in Set 1
SECTION: Day 3: Run the assay
33. Follow the sheet, add Tris buffer (5 mM ,pH 8.0) to each tube in Set 2. The unit of volume is uL.
   
SECTION: Day 3: Run the assay
34. Follow the sheet, add Tris buffer (5 mM ,pH 8.0) to each tube in Set 3. The unit of volume is uL.

   
SECTION: Day 3: Run the assay
35. Prepare STEB (0.05 % ) 
Add 500 µL STEB (1 % ) to 0.05% STEB tube, and vortex.
SECTION: Day 3: Run the assay
36. Add 250 µL STEB (0.05 % ) to each tube in Set 2.
SECTION: Day 3: Run the assay
37. Place RNase and DNase primary stock solutions, RNA and DNA primary standard solutions and samples on ice.
SECTION: Day 3: Run the assay
38. Turn on refrigerated centrifuge and set the temperature to 4 °C.
 

SECTION: Day 3: Run the assay
39. Turn on shaker/incubator and set temperature to 37 °C.
 

SECTION: Day 3: Run the assay
40. Prepare 900 mM MgCl2
SECTION: Day 3: Run the assay
40.1. Pour 1 M MgCl2 solution into 5 mL RNase free Falcon tube
 

SECTION: Day 3: Run the assay
40.2. Transfer 180 µL 1 M MgCl2 solution into 900 mM MgCl2 tube
SECTION: Day 3: Run the assay
41. Add 60 µL  900 mM MgCl2 to WS-A and WS-B
SECTION: Day 3: Run the assay
42. Prepare  900 mM  CaCl2
SECTION: Day 3: Run the assay
42.1. Pour 1 M CaCl2 solution into 5 mL RNase free Falcon tube
 

SECTION: Day 3: Run the assay
42.2. Transfer 180 µL 1 M CaCl2 solution into 900 mM CaCl2 tube
SECTION: Day 3: Run the assay
43. Add 60 µL  900 mM  CaCl2 to WS-A and WS-B
SECTION: Day 3: Run the assay
44. Prepare RNase working solution 0.5 mg/ml 
Add 10 µL RNase primary stock solution (10 mg/ml) to RNase tube 

SECTION: Day 3: Run the assay
45. Add 60 µL  0.5 mg/ml  RNase to WS-B and WS-C.
Keep WS-B and WS-C on ice .
SECTION: Day 3: Run the assay
46. Add 60 µL DNase primary stock solution ( 5 mg/ml ) to WS-A.
Keep WS-A on ice.
SECTION: Day 3: Run the assay
47. Centrifuge extracted samples  10000 x g, 4 min, 4 °C 
SECTION: Day 3: Run the assay
48. Prepare RNA secondary standard solution 2 ug/ml 
Add 15 µL RNA primary standard solution to RNA standard tube and vortex. 
Keep on ice .
SECTION: Day 3: Run the assay
49. Prepare DNA secondary standard solution 1 ug/ml 
Add 2 µL DNA primary standard solution to DNA standard tube and vortex. 
Keep on ice.
SECTION: Day 3: Run the assay
50. Load 50 µL WS-A to Tubes A in Set 2 and Set 3.
SECTION: Day 3: Run the assay
51. Load 50 µL WS-A to Tubes C in Set 2 and Set 3.
SECTION: Day 3: Run the assay
52. Load 50 µL WS-B to Tubes B in Set 2 and Set 3.
SECTION: Day 3: Run the assay
53. Load 50 µL WS-C to Tubes C in Set 2 and Set 3.
SECTION: Day 3: Run the assay
54. Add 100 µL centrifuged samples to its corresponding tubes in Set 1. 
Vortex each tube.
SECTION: Day 3: Run the assay
55. From Set 1, transfer 250 µL of diluted samples to each corresponding tubes in Set 3.
SECTION: Day 3: Run the assay
56. Follow the sheet:
Add RNA secondary standard to tubes (marked in pink) in Set 2.
Add  DNA secondary standard to tubes (marked in blue) in Set 2. 
The unit of volume is uL.

   
SECTION: Day 3: Run the assay
57. Vortex each tube for 2 s and place all tubes into the shaker/incubator at 37 °C, continuously shaking at 200 RPM for 20 min.
SECTION: Read fluorescence
58. Prepare SYBR Green II working solution (SG-II WS)
SECTION: Read fluorescence
58.1. Each 96-well microplate requires 1 mL of SG-II WS.
SECTION: Read fluorescence
58.2. Wrap 5 mL tube with foil, add 7 µL SYBR Green II 10,000X concentrate to each one mL Tris buffer (5 mM ,pH 8.0) in biosafety cabinet.
 

SECTION: Read fluorescence
58.3. Load 10 µL SG-II WS to each well in the microplate with 0.5 mL tip of stepper.
 

 

 

SECTION: Read fluorescence
58.4. Adhere black film on the top of a microplate lid and cover the plate with this lid.
 

 

SECTION: Read fluorescence
59. After incubation, vortex each tube for 2 s and then place into the fridge to stop the reaction.
SECTION: Read fluorescence
60. Allow samples to reach Room temperature for 2 min before loading the microplate.
SECTION: Read fluorescence
61. Load 190 µL working sample to the microplate. 
Standards must be included in each microplate.
Pink area: RNA standard solutions for RNA standard curves
Blue area: DNA standard solutions for DNA standard curves
Yellow area: Samples and blanks

   

 

SECTION: Read fluorescence
62.  Shake black film covered microplate at 37 Room temperature for 10 min
SECTION: Read fluorescence
63. Setup microplate reader:
Plate: Greiner F bottom chimney well PP 96 well;
Shake: Continuous 5s at 600 rpm
Endpoint reading: Ex 490 nm/Em 520 nm;
 

SECTION: Read fluorescence
64. Read fluorescence and export data to excel sheet.
SECTION: Calculate
65. RNA standard curve
SECTION: Calculate
65.1. Concentrations of RNA standards in the microplate
  
SECTION: Calculate
65.2. Slope of fluorescence in Tube A vs concentration of RNA standard gives mRNA+DNase 
Slope of fluorescence in Tube B vs concentration of RNA standard gives mRNA+RNase
SECTION: Calculate
65.3. Calculate ρ
  
SECTION: Calculate
66. 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
67. DNA standard curve 
SECTION: Calculate
67.1. Concentrations of DNA standards in the microplate
  
SECTION: Calculate
67.2. Slope of fluorescence in Tube A vs concentration of DNA standard gives mDNA+DNase 
Slope of fluorescence in Tube B vs concentration of DNA standard gives mDNA+RNase 
SECTION: Calculate
67.3. Calculate δ
  
SECTION: Calculate
68. 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
69. 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