TITLE: DNA/RNA extraction from fresh-frozen tissue, AllPrep DNA/RNA/miRNA Universal Kit
AUTHORS: Annika Fendler

[DESCRIPTION]
Protocol for combined RNA and DNA extraction from fresh-frozen tissue using the AllPrep DNA/RNA/miRNA Universal Kit.

[BEFORE_START]
Preparations:
FRN buffer: Add 42 ml Isoprop to new bottle
RPE buffer: Add 44 ml EtOH to new bottle
AW1 buffer: Add 25 ml EtOH to new bottle
AW2 buffer: Add 30 ml EtOH
DNAse I stocks: 550 µl RNase-free water to lyophilised DNAse I, aliquot and store at -20°C for 9 months)



[GUIDELINES]
DNase I stocks can be used 4 weeks after being thawed but should not be frozen again.
Never thaw tissue when processing.
Always use a fresh scalpel and plate when cutting tissues to prevent cross contamination.

[STEPS]
SECTION: Tissue preparation
4. This protocol is for   


SECTION: Tissue preparation
6. Transfer tissue in 350 µL 600 µL  RLT + ß-ME in 2 ml DNA LoBind tube.

SECTION: Tissue preparation
7. Add a 5mm bead to each tube and lyse tissue in  TissueLyser for 2 min  @ 20 Hz
 


SECTION: Tissue preparation
8. Spin down for 1 min @ 9000 x g 

SECTION: Tissue preparation
9. Turn tube rack and lyse tissue for 2 min  @ 20 Hz

SECTION: RNA extraction
17. Incubate 10 min @ Room temperature 

SECTION: Tissue preparation
10. Spin down for 1 min  @ 9000 x g 

SECTION: Tissue preparation
11. Add lysed product to DNA Mini Spin Column
SECTION: RNA extraction
15. Add 50 µL 80 µL  Proteinase K (not diluted), mix by pipetting

SECTION: RNA extraction
16. Add 200 µL  350 µL EtOH abs., mix by inverting, spin down liquid

SECTION: Tissue preparation
12. Spin 30 s @ max x g , repeat if any liquid remains on column

SECTION: Tissue preparation
13. Transfer column to a new collection tube and store tube at 4 °Cuntil DNA extraction

SECTION: Tissue preparation
14. Transfer flow-through to new 2 ml LoBind tube
SECTION: RNA extraction
18. Add 400 µL 750 µL EtOH abs. , mix by pipetting

SECTION: RNA extraction
19. Add 700 µL to RNeasy spin column and spin for 30 s @ max x g  and discard flow-through

SECTION: RNA extraction
20. Repeat until all liquid passed through the column
SECTION: RNA extraction
21. Add 500 µL RPE

SECTION: RNA extraction
22. Spin 30 s  @ max x g  and discard flow-through

SECTION: RNA extraction
23. Add 80 µL DNase I working solution directly onto the membrane and incubate 15 min at Room temperature 

SECTION: RNA extraction
24. Add500 µL FRN

SECTION: RNA extraction
25. Spin 30 s  @ max x g and don't discard flow-through and place column in new collection tube

SECTION: RNA extraction
26. Add flow-through again to column 
SECTION: RNA extraction
27. Spin 30 s @ max x g  and discard flow-through

SECTION: RNA extraction
28. Add 500 µL RPE

SECTION: RNA extraction
29. Spin 30 s @ max x g  and discard flow-through

SECTION: RNA extraction
30. Add 500 µLEtOH abs

SECTION: RNA extraction
31. Spin 2 min @ max x g  and place column in new collection tube

SECTION: RNA extraction
32. Spin 2 min @ max x g  and place column in new 1.5 ml collection tube

SECTION: RNA extraction
33. Add 30 µLof RNase-free H2O

SECTION: RNA extraction
34. Incubate 1 min @ Room temperature 

SECTION: RNA extraction
35. Spin down1 min @ 8000 x g 

SECTION: RNA extraction
36. QC: Qubit    and tape station   

56. 
SECTION: DNA extraction
37. 350 µL AW1 auf DNA Mini Spin Column geben

SECTION: DNA extraction
38. Spin 30 s @ max x g  and discard flow-through

SECTION: DNA extraction
39. 80 µLProteinase K working solution directly onto membrane

SECTION: DNA extraction
40. Incubate 5 min @ Room temperature 

SECTION: DNA extraction
41. Add 350 µL  AW1

SECTION: DNA extraction
42. Spin 30 s @ max x g  and discard flow-through

SECTION: DNA extraction
43. Add 350 µL AW2

SECTION: DNA extraction
44. Spin 2 min  @ max x g  and place column in new 2 ml collection tube

SECTION: DNA extraction
45. Spin 1 min  @ max x g  and place column in new 1.5 ml collection tube

SECTION: DNA extraction
46. Add 50 µL    directly onto membrane

SECTION: DNA extraction
47. Incubate 1 min  @ Room temperature

SECTION: DNA extraction
48. Spin down 1 min  @ 8000 x g 

SECTION: DNA extraction
49. QC: Qubit DNA    and tape station   

SECTION: RNA extraction
36.1. 



 


SECTION: DNA extraction
49.1. 


 

SECTION: Prepare Working Solutions
1. From    

DNAse I working solution: 70 µL RDD + 10 µL  DNase I working solution per sample
SECTION: Tissue preparation
4.1. Optional: Weigh tissue before start to decide for volume
SECTION: Tissue preparation
4.2. Enter a complete list of samples used for each experiment below:

 
 
 


SECTION: Prepare Working Solutions
2. Included within   

Proteinase K working solution for DNA isolation: 60 µL  AW1 + 20 µL  Proteinase K per sample

SECTION: Tissue preparation
5. Keep tubes with tissue on dry ice and make sure that they do not thaw during processing.
Transfer the tissue piece into a 6-well plate or small petry dish placed on dry ice and cut an appropriate piece of tissue (ca. 10-30 mg) with a safety scalpel.
SECTION: Prepare Working Solutions
3. Included within   
Add 10 µL ß-Mercaptoethanol (not included in kit) per 1 mL of RLT plus buffer