RNA Extraction Protocol

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Line 4: Line 4:
''Change gloves often to prevent ribonuclease contamination''
''Change gloves often to prevent ribonuclease contamination''
-
 
-
''Use fume hood when working with phenol!''
 
''See bottom of page for modifications for environmental/Lake Washington sediment modifications''
''See bottom of page for modifications for environmental/Lake Washington sediment modifications''
Line 30: Line 28:
:::*35 μl of SDS (20%) and 35 μl of sodium lauryl sarkosine (20%)
:::*35 μl of SDS (20%) and 35 μl of sodium lauryl sarkosine (20%)
:::*750 μl of phenol:chloroform:isoamylic alcohol (25:24:1)
:::*750 μl of phenol:chloroform:isoamylic alcohol (25:24:1)
-
:''Prepare 2 tubes per batch-grown sample, 9-12 tubes per sediment sample''
+
:''Prepare 2 tubes per batch-grown sample''
5. Remove samples from centrifuge.  '''''Keep on ice!'''''   
5. Remove samples from centrifuge.  '''''Keep on ice!'''''   
6. Discard the supernatant fraction and '''add 1 ml of extraction buffer''' to the pellet  
6. Discard the supernatant fraction and '''add 1 ml of extraction buffer''' to the pellet  
-
::*If extracting RNA from sediment samples, add 5 ml to be able to resuspend soil pellet
 
::*(''extraction buffer = ''[10% CTAB in 2.5 M NaCl]:0.1 M phosphate buffer pH 5.8, 1:3)
::*(''extraction buffer = ''[10% CTAB in 2.5 M NaCl]:0.1 M phosphate buffer pH 5.8, 1:3)
:Mix well and transfer 1 ml aliquots into prepared 2 ml screw-cap tubes   
:Mix well and transfer 1 ml aliquots into prepared 2 ml screw-cap tubes   
Line 48: Line 45:
10. Centrifuge at 14000 rpm for 5 min at 4°C.
10. Centrifuge at 14000 rpm for 5 min at 4°C.
:'''While centrifuging''', prepare tubes for precipitation (these will go in -80°C freezer)
:'''While centrifuging''', prepare tubes for precipitation (these will go in -80°C freezer)
-
::*5 ul 0.5M MgCl2
+
::*5 μL 0.5M MgCl2
-
::*75 ul 3M sodium acetate
+
::*75 μL 3M sodium acetate
11. Transfer supernatant to prepared tubes and add 800µL ice-cold isopropanol  
11. Transfer supernatant to prepared tubes and add 800µL ice-cold isopropanol  
Line 58: Line 55:
14. Centrifuge frozen tubes at 14,000 rpm (18,000 g) for '''1 hour''' at 4°C
14. Centrifuge frozen tubes at 14,000 rpm (18,000 g) for '''1 hour''' at 4°C
-
:Take Qiagen DNase I (aliquots in eppindorf tubes), Ambion DNase I (red cap) and buffer out of freezer and put on ice to thaw
+
:Take Qiagen DNase I (aliquots in eppindorf tubes), Ambion DNase I (red cap), Ambion buffer, and glycogen out of freezer and put on ice to thaw
15. Remove supernatant and '''add 500 μl of 75% ethanol'''
15. Remove supernatant and '''add 500 μl of 75% ethanol'''
Line 70: Line 67:
19. Dry samples for '''15 min''' at RT with caps open (evaporating alcohol)
19. Dry samples for '''15 min''' at RT with caps open (evaporating alcohol)
-
20. Pool every 2 samples (2-3 for sediment samples, 1 otherwise):  
+
20. Pool every 2 samples and:  
:Re-suspend in 100 μl of Ambion DNase I mix per pooled sample
:Re-suspend in 100 μl of Ambion DNase I mix per pooled sample
::Ambion DNase master mix:
::Ambion DNase master mix:
-
::*90 ul ddH2O
+
::*90 μL ddH2O
-
::*10 ul Ambion 10xDNase buffer
+
::*10 μL Ambion 10xDNase buffer
-
::*5 ul DNase I enzyme
+
::*5 μL DNase I enzyme
21. Incubate at 37°C  for '''30 min'''
21. Incubate at 37°C  for '''30 min'''
-
22. Add 350 μl Buffer RLT (from QIAGEN RNeasy kit)
+
22. '''Add 350 μl Buffer RLT''' (from QIAGEN RNeasy kit)
 +
 
 +
23. '''Add 250 ul ethanol and mix by pipetting
 +
::*Apply sample to an RNeasy mini column and centrifuge for 30 sec at 10,000 rpm (>8000g)
 +
::*Discard the flow-through
 +
 
 +
24. '''Add 500 μl RNeasy Buffer RW1''' to the RNeasy mini column and centrifuge for 15 sec at 10,000 rpm
 +
::Discard the flow-through '''and''' collection tube
 +
 
 +
25. Prepare and add 80 ul DNase I mix per column
 +
::Qiagen DNase master mix:
 +
::*70 ul RNeasy buffer RDD
 +
::*10 ul DNase I enzyme
 +
 
 +
26. '''Add 500 μl RNeasy Buffer RW1''' to the column and centrifuge for 30 sec at 10,000 rpm
 +
::Discard the flow-through '''and''' collection tube
 +
 
 +
27. Transfer the RNeasy mini column into a new collection tube
 +
 
 +
28. '''Add 500 μl RNeasy Buffer RPE''' and centrifuge for 15 sec at 10,000 rpm
 +
::Discard the flow-through
 +
 
 +
29. '''Add 500 μl RNeasy Buffer RPE''' and centrifuge for 15 sec at 10,000 rpm
 +
::Discard the flow-through
 +
 
 +
30. '''To eliminate any chance of possible Buffer RPE carryout,''' place the RNeasy mini column in a new 1.5 ml tube and centrifuge for 1 min at 10,000 rpm
 +
 
 +
31. Transfer RNeasy mini column into a new 1.5 ml tube
 +
 
 +
32. Add 35 (or 50 to be quite sure) μl of sterile DNase-, RNase-free water '''to the membrane''' (be sure that the elution water doesn't stick to side of tube) and centrifuge for 1 min at 10,000 rpm
 +
 
 +
'''''DO NOT DISCARD THE FLOW-THROUGH''''' 
 +
 
 +
33. Add 35 μl of sterile DNase RNase-free water, '''wait 2 minutes''' then centrifuge for 1 min at 10,000 rpm 
 +
 
 +
34. Combine several effluents together if applicable
 +
 
 +
35. Check RNA for quality and quantity by NanoDrop and electrophoresis (1% agarose gel)
 +
::*'''If RNA quality is good''', take out 10 μL before precipitation to use on Bioanalyzer chip or for RT-qPCR
 +
::*'''If RNA quality is bad''', a second round of Day 2 clean up can be performed after re-precipitating at -80°C overnight
 +
:::*At step 21, incubate at room temperature and NOT at 37°C
 +
 
 +
36. Re-precipitate isolated RNA by adding appropriate amount of glycogen, sodium acetate & ethanol:
 +
:::*3 volumes ethanol
 +
:::*1/10 original volume of sodium acetate
 +
:::*1/50 original volume of glycogen
 +
 
 +
==Storage of RNA==
 +
- Small aliquots for analysis are fine frozen as liquid
 +
 
 +
- For longer-term storage, larger volumes of RNA can be:
 +
*Stored in re-precipitation stage for up to a week
 +
*Stored as a dry pellet prepared by:
 +
:::- Centrifuge precipitated tube at 4°C at 14,000 rpm for 1 hour
 +
:::- Remove supernatant
 +
:::- Add 100 ul 70% EtOH
 +
:::- Centrifuge at 4°C at 14,000 rpm for 5 min
 +
:::- Remove supernatant and allow pellet to dry for 15 min at room temp
 +
:::- Store at -80°C
 +
 
 +
 
 +
==Environmental/Lake Washington sediment modifications==
 +
Day 1 modifications:
 +
*At step 4: Prepare 9-12 tubes per sediment sample
 +
*At step 6: Increase lysis buffer to 5 ml to be able to resuspend soil pellet
-
23. For LWS pooled samples, do the following twice:
+
Day 2 modifications:
 +
*At step 20: Pool every 2-3 samples
 +
*At step 21: If you are on your second cleanup, incubate '''at room temperature''', not 37°C
 +
*At step 23: Perform two times

Revision as of 18:29, 22 May 2013

Extraction Protocol modified from Griffiths et. al, 2000

Can also be used for DNA extraction by increasing phosphate buffer pH to ~8

Change gloves often to prevent ribonuclease contamination

See bottom of page for modifications for environmental/Lake Washington sediment modifications


This protocol uses:

  • Ambion DNase I [1]
  • Qiagen DNase I [2] prepared as directed and divided into tubes containing 50-70 ul each (prevents contamination and freeze/thaws of entire supply)
  • Qiagen RNeasy Mini Kit [3]

Contents

Day 1

1. Start centrifuge cooling with 50 mL tube holders

2. Add 5 ml of cold stop solution to a fresh 50 mL tube

(stop solution = 5% buffer equilibrated phenol [pH 7.4] in ethanol)

3. Add sample up to 50 ml

4. Pellet sample by centrifugation at 5000 rpm for 10 min at 4°C

While centrifuging, prepare 2 ml screw-cap tubes with:
  • 0.5 g of 0.1 mm zirconia-silica beads (Biospec products)
  • 35 μl of SDS (20%) and 35 μl of sodium lauryl sarkosine (20%)
  • 750 μl of phenol:chloroform:isoamylic alcohol (25:24:1)
Prepare 2 tubes per batch-grown sample

5. Remove samples from centrifuge. Keep on ice!

6. Discard the supernatant fraction and add 1 ml of extraction buffer to the pellet

  • (extraction buffer = [10% CTAB in 2.5 M NaCl]:0.1 M phosphate buffer pH 5.8, 1:3)
Mix well and transfer 1 ml aliquots into prepared 2 ml screw-cap tubes

7. Homogenise in a mini-beater (Biospec products) for 2 min (3 min in less powerful beater)

8. Centrifuge at 14000 rpm for 5 min at 4°C

9. Transfer the upper aqueous phase into a new 2 ml tube

Add 750 µl chloroform:isoamylic alcohol (24:1)

10. Centrifuge at 14000 rpm for 5 min at 4°C.

While centrifuging, prepare tubes for precipitation (these will go in -80°C freezer)
  • 5 μL 0.5M MgCl2
  • 75 μL 3M sodium acetate

11. Transfer supernatant to prepared tubes and add 800µL ice-cold isopropanol

12. Incubate overnight at -80°C to allow full precipitation of nucleic acids.

Day 2

13. Cool RNA room centrifuge

14. Centrifuge frozen tubes at 14,000 rpm (18,000 g) for 1 hour at 4°C

Take Qiagen DNase I (aliquots in eppindorf tubes), Ambion DNase I (red cap), Ambion buffer, and glycogen out of freezer and put on ice to thaw

15. Remove supernatant and add 500 μl of 75% ethanol

16. Centrifuge at 14,000 rpm for 5 min at 4°C

17. Remove supernatant and centrifuge at 14,000 rpm for 2 min at 4°C

18. Remove any liquid residue from the tubes by pipetting

19. Dry samples for 15 min at RT with caps open (evaporating alcohol)

20. Pool every 2 samples and:

Re-suspend in 100 μl of Ambion DNase I mix per pooled sample
Ambion DNase master mix:
  • 90 μL ddH2O
  • 10 μL Ambion 10xDNase buffer
  • 5 μL DNase I enzyme

21. Incubate at 37°C for 30 min

22. Add 350 μl Buffer RLT (from QIAGEN RNeasy kit)

23. Add 250 ul ethanol and mix by pipetting

  • Apply sample to an RNeasy mini column and centrifuge for 30 sec at 10,000 rpm (>8000g)
  • Discard the flow-through

24. Add 500 μl RNeasy Buffer RW1 to the RNeasy mini column and centrifuge for 15 sec at 10,000 rpm

Discard the flow-through and collection tube

25. Prepare and add 80 ul DNase I mix per column

Qiagen DNase master mix:
  • 70 ul RNeasy buffer RDD
  • 10 ul DNase I enzyme

26. Add 500 μl RNeasy Buffer RW1 to the column and centrifuge for 30 sec at 10,000 rpm

Discard the flow-through and collection tube

27. Transfer the RNeasy mini column into a new collection tube

28. Add 500 μl RNeasy Buffer RPE and centrifuge for 15 sec at 10,000 rpm

Discard the flow-through

29. Add 500 μl RNeasy Buffer RPE and centrifuge for 15 sec at 10,000 rpm

Discard the flow-through

30. To eliminate any chance of possible Buffer RPE carryout, place the RNeasy mini column in a new 1.5 ml tube and centrifuge for 1 min at 10,000 rpm

31. Transfer RNeasy mini column into a new 1.5 ml tube

32. Add 35 (or 50 to be quite sure) μl of sterile DNase-, RNase-free water to the membrane (be sure that the elution water doesn't stick to side of tube) and centrifuge for 1 min at 10,000 rpm

DO NOT DISCARD THE FLOW-THROUGH

33. Add 35 μl of sterile DNase RNase-free water, wait 2 minutes then centrifuge for 1 min at 10,000 rpm

34. Combine several effluents together if applicable

35. Check RNA for quality and quantity by NanoDrop and electrophoresis (1% agarose gel)

  • If RNA quality is good, take out 10 μL before precipitation to use on Bioanalyzer chip or for RT-qPCR
  • If RNA quality is bad, a second round of Day 2 clean up can be performed after re-precipitating at -80°C overnight
  • At step 21, incubate at room temperature and NOT at 37°C

36. Re-precipitate isolated RNA by adding appropriate amount of glycogen, sodium acetate & ethanol:

  • 3 volumes ethanol
  • 1/10 original volume of sodium acetate
  • 1/50 original volume of glycogen

Storage of RNA

- Small aliquots for analysis are fine frozen as liquid

- For longer-term storage, larger volumes of RNA can be:

  • Stored in re-precipitation stage for up to a week
  • Stored as a dry pellet prepared by:
- Centrifuge precipitated tube at 4°C at 14,000 rpm for 1 hour
- Remove supernatant
- Add 100 ul 70% EtOH
- Centrifuge at 4°C at 14,000 rpm for 5 min
- Remove supernatant and allow pellet to dry for 15 min at room temp
- Store at -80°C


Environmental/Lake Washington sediment modifications

Day 1 modifications:

  • At step 4: Prepare 9-12 tubes per sediment sample
  • At step 6: Increase lysis buffer to 5 ml to be able to resuspend soil pellet

Day 2 modifications:

  • At step 20: Pool every 2-3 samples
  • At step 21: If you are on your second cleanup, incubate at room temperature, not 37°C
  • At step 23: Perform two times
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