WASHING _volume 500 ml for 8 arrays_

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					MicroRNA isolation and array hybridization                                      Subramanian 2006

I. Total RNA Isolation Protocol ( miRvana 1560)

1.Measure or estimate the weight of the tissue sample. 150-200mg sample is optimal
2. Aliquot 3ml of Lysis/Binding Buffer into a homogenization tube on ice
3. Homogenate the sample for 2 mins and leave on ice till all samples are completed
4. Add 300 μl of miRNA homogenate additive to the lysate, vortex 30sec.
5. Leave the mixture on ice for 10 min
6. Add 3 ml of Acid –Phenol: chloroform to the mixture
7. Vortex for 30-60 sec
8. Centrifuge for 15 min at 11000 rpm
9. transfer the aqueous phase to a fresh tube (~2.5 to 3ml).
10. Add 1.25 volume of 100% Ethanol (3.75 ml)
11. Assemble filter cartridge and collection tube. Pass the lysate/ethanol mixture through a Filter
12. Up to 700 μl can be applied to a Filter Cartridge at a time, for samples larger than this, apply
the mixture in successive applications to the same filter
13. Centrifuge at 10,000 x g for ~15 sec to pass the mixture through the filter.
14. Discard the flow-through, and repeat until all of the lysate/ethanol mixture is over
15. Apply 700 μl miRNA Wash Solution 1 (working solution mixed with
ethanol) to the Filter Cartridge and centrifuge for ~5–10 sec
16. Apply 500 μl Wash Solution 2/3 (working solution mixed with ethanol) and draw it through
the Filter Cartridge as in the previous step
17. Repeat with a second 500 μl aliquot of Wash Solution 2/3
18. After discarding the flow-through from the last wash, replace the Filter Cartridge in the same
Collection Tube and spin the assembly for 1 min to remove residual fluid from the filter
19. Elute RNA with 125 μl 95°C Elution Solution or Nuclease-free Water
20. Spin for 1 min at maximum speed to recover the RNA.

II. Check OD and quality of RNA

MicroRNA isolation and array hybridization                              Subramanian 2006

miRNA isolation from total RNA

1. Aliquot 25 µg total RNA in 5 mM Tris pH7.6 (sample+ Tris buffer should not exceed 50-60µl
2. Apply sample to YM-100 microcon column, and spin 6000 rpm 3min at RT
3. Save the eluate (miRNAs), keep on ice before labeling or store at -20/-80C
5. Recover the left over total RNA sample by reversing the column (on a collection tube) and
apply 10 µl 1mM Tris pH7.6 and spin 3min 1,000g at RT.

6. Dry the miRNA sample to completion in a vacuum drier

miRNA labeling (follow Ambion 1562 kit procedure).
1. Resuspend miRNA samples in 3 μl Nuclease-free Water

2. Add Poly(A) Polymerase reaction mix to each miRNA sample

 1 μl   diluted Positive (1:100) Control miRNA
10 μl   2X Poly(A) Polymerase Reaction Buffer
 2 μl   25 mM MnCl2
 2 μl   10X Amine-NTP Mix
 2 μl   Poly(A) Polymerase

3. Incubate at 37°C for 2 hr

Post-Tailing miRNA Clean-Up

1. To each reaction add the following:

10 μl miRNA Carrier
350 μl miRNA Binding/Wash Buffer

2. Mix by vortexing briefly, then incubate at room temp for 5 min.

3. Wash with 2 x 300 μl miRNA Binding/Wash Buffer

4. Dry the filter by centrifuging at ~10,000 x g for 1 min.

5. Elute tailed miRNA with 30 μl 95c hot Elution Solution

6. Dry samples in a vacuum concentrator

        note: dried sample can be store for 1-2 day in -20C

MicroRNA isolation and array hybridization                                    Subramanian 2006

miRNA slides post processing

I.      Mark the edges of the spot printed area on back side of the slide.

II.     Hydration and immobilization
Incubate printed microarray slides in humidity chamber (0.5X SSC @42C) for 30 min.

III.   WASHING (volume 500 ml for 8 arrays)
Wash slides to remove unbound probe molecules and buffer substances to avoid interference with
subsequent hybridization experiments.

a. Rinse 1 x 5 min in 0.1% Triton X-100 at room temperature.
b. Rinse 2 x 2 min in 1 mM HCl solution at room temperature.
c. Rinse 1 x 10 min in 100 mM KCl solution at room temperature.
e. Rinse 1 x 1 min in diH2O at room temperature.

Proceed to Blocking immediately.

IV.     BLOCKING (volume 500 ml for 8 arrays)

50 mM ethanolamine, 0.1% SDS (add freshly before use) in 0.1 M Tris, pH 9.0

1. Incubate slides 1 x 15 min in Blocking Solution at 50°C.
2. Rinse 1 x 1 min in diH2O at room temperature.
3. Dry by centrifugation (200 x g for 5 min) to avoid any water stains on the slide surface.

V. Labeling the Tailed miRNA with Amine-Reactive CyDye

1. Resuspend in 7 μl Nuclease-free Water

2. Resuspend Cy3- or Cy5-NHS ester (Amersham # RPN5661) with 16 μl of DMSO

3. Add 9 μl Coupling Buffer and 4 μl prepared CyDye to each sample

4. Vortex briefly to mix, and shield from light as much as possible.

5. Incubate 1 hr at room temp in the dark

6. Add 4.5 μl 4M Hydroxylamine and mix

7. Incubate 15 min at room temp in the dark

VI. Post-Labeling miRNA Clean-Up

1. Add 350 μl miRNA Binding/Wash Buffer and incubate 5 min at room temp in the dark

2. Pass mixture through a miRNA labeling cartridge

MicroRNA isolation and array hybridization                                 Subramanian 2006

3. Wash with 2 x 300 μl miRNA binding/Wash buffer

4. Dry the filter by centrifuging at ~10,000 x g for 1 min.

5. Add 22 μl of 95°C nuclease-free water to the cartridge and incubate at 65°C for 10 min

6. Centrifuge at 10000 g for 1 min

Immediately proceed to array hybridization

22 μl labeled product +10μl hyb buffer (#8860G)
Heat the mixture at 95C for 3 min and cool to RT.
Keep post processed slides warm at 37 prior to hyb (optional)
Use lifter slips (22X40mm)
Hyb at 42c for 12-16 hrs

Washing (solutions from ambion # 9823G5 and 9763G6)
1. Use 1X low wash for 1 min
        20 ml salt conc
        4 ml detergent conc make up to 400 ml with dH2O

2. 2X high stringency wash for 1 min each
        2 ml salt conc in 398 ml dH2O
3. Dry the slides by centrifugation at 500 rpm for 5 min

Scan the arrays immediately.


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