TITLE: RNA Isolation from QIAzol sample with miRNeasy Mini Kit
AUTHORS: Stephan Schreiber

[DESCRIPTION]
__________________________________________________________________________
Purpose
 
This SOP describes the procedure of total RNAextraction from cell and tissue samples using the Qiagen miRNeasy Mini kit and Ambion Turbo DNA-free kit. The miRNeasy Mini Kit combines phenol/guanidine-based lysis of samples and silica–membrane-based purification of total RNA. QIAzol Lysis Reagent, included in the kit, is a monophasic solution of phenol and guanidine thiocyanate designed to facilitate the lysis of tissues, inhibit RNases, and remove most of the cellular DNA and proteins from the lysate by organic extraction. Cells or tissue samples are homogenized in the QIAzol Lysis reagent. After adding chloroform, centrifugation separates the homogenate into aqueous and organic phases. RNA partitions to the upper, aqueous phase, while DNA partitions to the interphase and proteins to the lower, organic phase or the interphase. The upper aqueous phase is extracted, and ethanol is added to provide appropriate binding conditions for all RNA molecules from 18 nucleotides (nt) upwards. The sample is then applied to the RNeasy Mini spin column, where the total RNA binds to the membrane, and phenol and other contaminants are efficiently washed away. High-quality RNA is then eluted in RNase-free water. Eluted RNA is treated with an Ambion TURBO DNA-free kit to remove contaminating DNA. A final cleanup step by Ethanol precipitation removes traces of DNase buffer and enzyme to avoid effects on downstream applications, like reverse transcription of RNA into cDNA.
 
This protocol extracts the following classes of RNA: total RNA (including mRNA, miRNA, rRNA, tRNA, snRNA, snoRNA, piRNA and lncRNA)


[BEFORE_START]
Each surface must be decontaminated with RNase AWAY beforehand. All pipetting steps must be performed under RNase-free conditions.

[GUIDELINES]
For Section-A: Steps are performed under the fume hood
For Section-B: Steps are performed under the PCR workbench
For Section-C: Steps are performed under the PCR workbench
For Section-D: Steps are performed under the PCR workbench

[STEPS]
SECTION: A.	Prepare sample and material (starting from 1 mL QIAzol sample)
1. Place the tube containing the sample (cell pellets or homogenized tissue in QIAzol reagent) on the bench top at room temperature (15-25 °C) for 5 min.

SECTION: A.	Prepare sample and material (starting from 1 mL QIAzol sample)
2. At this time, centrifuge the MaXtract tube at 14.000 x g for 30 s.

SECTION: A.	Prepare sample and material (starting from 1 mL QIAzol sample)
3. Pre-cool the centrifuge to 4 °C.

SECTION: A.	Prepare sample and material (starting from 1 mL QIAzol sample)
4. Mix QIAzol-sample by pipetting or choose another appropriate homogenization method.
SECTION: A.	Prepare sample and material (starting from 1 mL QIAzol sample)
5. Transfer by pipetting 1000 µL QIAzol-sample to the MaXtract® tube.

SECTION: A.	Prepare sample and material (starting from 1 mL QIAzol sample)
6. Add 200 µL chloroform.

SECTION: A.	Prepare sample and material (starting from 1 mL QIAzol sample)
7. Securely cap the tube and shake it vigorously for 15 s. DO NOT VORTEX!

SECTION: A.	Prepare sample and material (starting from 1 mL QIAzol sample)
8. Place the tube on the bench top at Room temperature for 3 min.

SECTION: A.	Prepare sample and material (starting from 1 mL QIAzol sample)
9. Centrifuge at 12.000 x g, 4 °C for 15 min.

SECTION: A.	Prepare sample and material (starting from 1 mL QIAzol sample)
10. In the meantime, label spin columns and 1.5 mL SafeLockTubes for ethanol mixture and elution (if necessary, prepare Buffer RWT and Buffer RPE as indicated in the protocol sheet “miRNeasy Mini”).
SECTION: A.	Prepare sample and material (starting from 1 mL QIAzol sample)
11. Pre-warm the centrifuge to20 °C.

SECTION: A.	Prepare sample and material (starting from 1 mL QIAzol sample)
12. Transfer the upper, aqueous phase from the Maxtract tube to a new 1.5 mL safe-lock tube (by decanting).
SECTION: A.	Prepare sample and material (starting from 1 mL QIAzol sample)
13. Add 750 µL (around 1.5 volumes) of 100 % (v/v) ethanol and mix thoroughly by pipetting.

SECTION: A.	Prepare sample and material (starting from 1 mL QIAzol sample)
14. Pipet 700 µL sample, including any precipitate, into an RNeasy Mini spin column in a 2 mL collection tube. Close the lid and centrifuge at room temperature at 8.000 x g for 30 s. Discard the flow-through.

SECTION: A.	Prepare sample and material (starting from 1 mL QIAzol sample)
15. Repeat the last step (A14) using the remainder of the sample.
SECTION: B.	Wash spin column and elute RNA
16. Add 700 µL Buffer RWT to the spin column. Close the lid and centrifuge for 30 s at 8.000 x g. Discard the flow-through.

SECTION: B.	Wash spin column and elute RNA
17. Add 500 µL Buffer RPE to the spin column. Close the lid and centrifuge for 30 s at 8.000 x g. Discard the flow-through.

SECTION: B.	Wash spin column and elute RNA
18. Add another 500 µL Buffer RPE to the spin column. Close the lid and centrifuge for 2 min at 8.000 x g. Discard the flow-through.

SECTION: B.	Wash spin column and elute RNA
19. Place the spin column into a new 2 ml collection tube and centrifuge at 12.000 x g for 1 min to dry the membrane further.

SECTION: B.	Wash spin column and elute RNA
20. Transfer the spin column to a new 1.5 mL SafeLockTube. Pipet 30 µL RNase-free water directly onto the spin column membrane. Close the lid and centrifuge for 1 min at 8.000 x g to elute.

SECTION: B.	Wash spin column and elute RNA
21. Remove and discard spin columns from elution tubes.
SECTION: B.	Wash spin column and elute RNA
22. Put elution tubes with total RNA on ice.

SECTION: C.	DNase digestion
23. Preheat the ThermoMixer with 0.5 mL block and ThermoTop at 37 °C.

SECTION: C.	DNase digestion
24. Prepare and label 0.5 mL SafeLockTubes.
SECTION: C.	DNase digestion
25. Add 3 µL 10x Turbo DNase Buffer to the samples.

SECTION: C.	DNase digestion
26. Add 1.5 µL Turbo DNase to the samples.

SECTION: C.	DNase digestion
27. Transfer samples to 0.5 mL SafeLockTubes and mix gently by pipetting.
SECTION: C.	DNase digestion
28. Incubate samples for 30 min at 37 °C.

SECTION: C.	DNase digestion
29. Take samples from the ThermoMixer and add another 1.5 µL Turbo DNase. Mix by pipetting.

SECTION: C.	DNase digestion
30. Incubate the samples for 30 min at 37 °C

SECTION: C.	DNase digestion
31. During this time, prepare and label 1.5 mL SafeLockTubes for digested total-RNA and thaw DNase Inactivation reagent.
SECTION: C.	DNase digestion
32. Remove the samples from the ThermoMixer and add 7.2 µL of well-mixed Turbo DNase Inactivation reagent. Mix by pipetting.

SECTION: C.	DNase digestion
33. Incubate for 5 min at room temperature, mixing occasionally by vortexing at  .

SECTION: C.	DNase digestion
34. Centrifuge at 10.000 x gfor90 s.

SECTION: C.	DNase digestion
35. Transfer as much RNA as possible (upper, aqueous phase) to 1.5 mL SafeLockTubes by pipetting. Don’t transfer some of the white sediment to the new tube.
SECTION: D.	RNA precipitation
36. Add 140 µL RNase-free water to the sample.

SECTION: D.	RNA precipitation
37. Add 4 µL Glycogen to the samples.

SECTION: D.	RNA precipitation
38. Add 20 µL 3M Sodium Acetate to the samples and mix by pipetting.

SECTION: D.	RNA precipitation
39. Add 600 µL of ice-cold 100% ethanol to each sample and mix by pipetting.

SECTION: D.	RNA precipitation
40. Place samples at -20 °C 5 min.

SECTION: D.	RNA precipitation
41. The next day: Pre-cool the centrifuge to 4 °C.

SECTION: D.	RNA precipitation
42. Centrifuge samples at 12.000 x g, 4 °C for 30 min.

SECTION: D.	RNA precipitation
43. Carefully remove and discard the supernatant.
SECTION: D.	RNA precipitation
44. Wash the pellet with 500 µL ice-cold 70 % (v/v) ethanol and vortex the solution briefly.

SECTION: D.	RNA precipitation
45. Centrifuge samples at7.600 x g, 4 °C for 5 min.

SECTION: D.	RNA precipitation
46. Carefully remove and discard the supernatant.
SECTION: D.	RNA precipitation
47. Wash the pellet with another 500 µL ice-cold 70 % (v/v) ethanol and vortex the solution briefly.

SECTION: D.	RNA precipitation
48. Centrifuge samples at 7.600 x g, 4 °C for 5 min.

SECTION: D.	RNA precipitation
49. Carefully remove and discard the supernatant.
SECTION: D.	RNA precipitation
50. Centrifuge samples at 7.600 x g, 4 °C for 30 s.

SECTION: D.	RNA precipitation
51. Carefully remove and discard any rest of the ethanol by pipetting.
SECTION: D.	RNA precipitation
52. Under the PCR workbench, with the tube lid open, allow the pellet to air dry at room temperature (do not overdry the pellet).
SECTION: D.	RNA precipitation
53. At this time, preheat the ThermoMixer with 1.5 mL block and ThermoTop at58 °C.

SECTION: D.	RNA precipitation
54. Dissolve the pellet in 20 µL of RNase-free water by pipetting up and down.

SECTION: D.	RNA precipitation
55. Incubate samples for 8 min at 58 °C in a ThermoMixer.

SECTION: D.	RNA precipitation
56. Place the samples on ice to cool down. Mix the samples by flicking. Centrifuge briefly to collect the entire sample.

SECTION: D.	RNA precipitation
57. Store total RNA at -80 °C.

SECTION: E.	Cleaning
58. Clean all used materials and put them back.
SECTION: E.	Cleaning
59. Empty all waste bins and discard collection of flow through.
SECTION: E.	Cleaning
60. Clean all used working areas with RNaseAWAY.
SECTION: E.	Cleaning
61. Turn off all used instruments.
SECTION: E.	Cleaning
62. Turn on the PCR workbench UV light for 30 minutes.