TITLE: RNA Extraction from Wastewater Concentrates Using RNeasy and Zymo Kits
AUTHORS: Jacquelina.Woods  , rachel.rodriguez  

[DESCRIPTION]
This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This method describes the extraction of RNA from viral concentrates using the RNeasy and Zymo kits. 

[BEFORE_START]
Heat Primer TE at 70 °C for at least 10 min prior to use.
 


 


[STEPS]
SECTION: RNeasy Mini
1. Obtain one virus concentrate (if concentrate is frozen, allow thawing) from Virus Concentration from Wastewater Using PEG Precipitation and Ultracentrifugation (protocols.io)



SECTION: RNeasy Mini
2. Add 500 µL 6M GITC.
 
.

SECTION: RNeasy Mini
3. Vortex 75 s to dissolve concentrate.


SECTION: RNeasy Mini
4. Add 700 µL of 50% EtOH and invert twice.

SECTION: RNeasy Mini
5. Pipette 700 µLof sample onto a RNeasy mini spin column.

SECTION: RNeasy Mini
6. Centrifuge 10000 x g, 1 min.

SECTION: RNeasy Mini
7. Place column in new collection tube and discard flow through.
SECTION: RNeasy Mini
8. Add remaining sample from Step 4 to column.
SECTION: RNeasy Mini
9. Centrifuge 10000 x g, 1 min.

SECTION: RNeasy Mini
10. Place column in new collection tube and discard flow through.
SECTION: RNeasy Mini
11. Add 700 µL RW1 buffer to spin column and incubate for 15 min at room temperature.

SECTION: RNeasy Mini
12. Centrifuge 10000 x g, 1 min.
SECTION: RNeasy Mini
13. Place column in new collection tube and discard flow through.
SECTION: RNeasy Mini
14. Add 500 µL RPE to spin column and incubate for 15 min.

SECTION: RNeasy Mini
15. Centrifuge 10000 x g, 1 min.
SECTION: RNeasy Mini
16. Add an additional 500 µL of RPE to Mini spin column.

 

SECTION: RNeasy Mini
17.    Centrifuge at maximum speed >16000 x g, 2 min. 

 

SECTION: RNeasy Mini
18. Transfer column to new collection tube.   
SECTION: RNeasy Mini
19.  Centrifuge 16000 x g, 1 min. 
SECTION: RNeasy Mini
20. Carefully transfer column to 1.5 ml low-retention/siliconized RNase/DNase free microcentrifuge tube.
SECTION: RNeasy Mini
21. Pipette 50 µL pre-heated (70°C) Primer TE onto silica-gel membrane of column. 
 


SECTION: RNeasy Mini
22.  Centrifuge 10000 x g, 1 min. 
 

SECTION: RNeasy Mini
23. Pipette an additional 50 µL pre-heated (70°C) Primer TE onto silica-gel membrane of column.
SECTION: RNeasy Mini
24. Pipette the eluted RNA (from Step 22) 50 µL back onto column.
SECTION: RNeasy Mini
25.  Centrifuge 10000 x g, 1 min. 
SECTION: RNeasy Mini
26. Discard column and place tube with RNA on on iceto prepare Zymo column.

SECTION: RNeasy Mini
27. Prepare Zymo column per manufacturer's instructions.
SECTION: RNeasy Mini
28. Transfer Zymo column into a clean 1.5 or 2.0 ml low-retention/siliconized RNase/DNase free microcentrifuge tube.
SECTION: RNeasy Mini
29. Transfer RNA from Step 26 to prepared Zymo One Step RT-PCR inhibitor remover column.
SECTION: RNeasy Mini
30. Centrifuge 8000 x g, 3 min. 

SECTION: RNeasy Mini
31. Discard column and save RNA in the 1.5 or 2.0 ml low-retention/siliconized RNase/DNase free microcentrifuge tube.
SECTION: RNeasy Mini
32. Proceed with RT-qPCR or store RNA at -70 °C. 

SECTION: RNeasy Midi
33. Obtain two virus concentrates (if concentrate is frozen, allow thawing) from Virus Concentration from Wastewater Using PEG Precipitation and Ultracentrifugation (protocols.io)

 



SECTION: RNeasy Midi
34. Optional chloroform clean-up. 
   

SECTION: RNeasy Midi
35. Add 2 mL 6M GITC. 
 

SECTION: RNeasy Midi
36. Vortex 75 s.

SECTION: RNeasy Midi
37. Add 2.8 mL of 50% EtOH and invert twice.

SECTION: RNeasy Midi
38. Pipette4 mL of sample onto a RNeasy Midi spin column.

SECTION: RNeasy Midi
39. Centrifuge 5000 x g, 5 min.

SECTION: RNeasy Midi
40. Discard flow through and return Midi column to tube.
SECTION: RNeasy Midi
41. Add remaining sample (from Step 40) to Midi column.
SECTION: RNeasy Midi
42. Centrifuge 5000 x g, 5 min.
SECTION: RNeasy Midi
43. Place column in new collection tube (sterile, nuclease-free 15 ml conical tube) and discard flow through.
SECTION: RNeasy Midi
44. Add4 mL RW1 buffer to Midi spin column. 

SECTION: RNeasy Midi
45. Incubate for 15 min atRoom temperature.

SECTION: RNeasy Midi
46. Centrifuge 5000 x g, 5 min.
SECTION: RNeasy Midi
47. Place column in new collection tube (sterile, nuclease-free 15 ml conical tube) and discard flow through.
SECTION: RNeasy Midi
48. Add 4 mL RPE to Midi spin column and incubate for 15 min at Room temperature. 

SECTION: RNeasy Midi
49. Centrifuge 5000 x g, 5 min.
SECTION: RNeasy Midi
50. Transfer column to new collection tube and add an additional 4 mLof RPE to the Midi spin column.

 

SECTION: RNeasy Midi
51.  Centrifuge 5000 x g, 10 min.
SECTION: RNeasy Midi
52. Carefully transfer column to a sterile, nuclease-free 15 ml conical tube.
SECTION: RNeasy Midi
53. Pipette 150 µLheated (70 °C) Primer TE onto silica-gel membrane of Midi column.

SECTION: RNeasy Midi
54. Incubate for1 min at 70 Room temperature.

SECTION: RNeasy Midi
55.  Centrifuge 5000 x g, 3 min.
 

SECTION: RNeasy Midi
56. Add an additional 150 µLof heated Primer TE onto the Midi column.

SECTION: RNeasy Midi
57. Pipette the eluted RNA (Step 55) back onto column (~150 µL).

SECTION: RNeasy Midi
58. Centrifuge 5000 x g, 3 min.
SECTION: RNeasy Midi
59. Discard column and place tube with RNA on ice to prepare Zymo column.

SECTION: RNeasy Midi
60. Prepare two (2) Zymo One Step RT-PCR inhibitor remover columns per manufacturer's instructions. 
SECTION: RNeasy Midi
61. Transfer Zymo column into a clean 1.5 or 2.0 ml low-retention/siliconized RNase/DNase free micriocentrifuge tube.
SECTION: RNeasy Midi
62. Transfer RNA from Step 59 to prepared Zymo One Step RT-PCR inhibitor remover columns (~150 µLeach) .

SECTION: RNeasy Midi
63. Centrifuge 8000 x g, 3 min.
SECTION: RNeasy Midi
64. Combine RNA from columns into one fresh low-retention/siliconized RNase/DNase free micriocentrifuge tube.
SECTION: RNeasy Midi
65. Proceed with RT-qPCR (RT-qPCR Detection of Process Controls (Murine noroviurs and crAssphage) from Wastewater (protocols.io) and RT-qPCR Detection of SARS-CoV-2 from Wastewater Using the AB 7500 (protocols.io)) or store at-70 °C .

SECTION: RNeasy Midi
34.1. Add 800 µLof ..
SECTION: RNeasy Midi
34.2. Vortex 45 s.
SECTION: RNeasy Midi
34.3. Centrifuge 3000 x g, 5 min.
SECTION: RNeasy Midi
34.4. Transfer aqueous layer to 5 ml Eppendorf low bind centrifuge tube.
SECTION: RNeasy Mini
27.1. Insert column into a collection tube. 
SECTION: RNeasy Mini
27.3. Centrifuge at 8000 x g, 3 min. 

SECTION: RNeasy Mini
27.2. Open the cap and add 600 µL of Prep-solution.

SECTION: RNeasy Midi
60.1. Insert Zymo column into a collection tube. 
SECTION: RNeasy Midi
60.3. Centrifuge at 8000 x g, 3 min. 
SECTION: RNeasy Midi
60.2. Open the cap and add 600 µL of Prep-solution.