RNA Extraction

Day 1

Grind 3-6g of Arabidopsis leaves in liquid N2 with mortar and pestle.

Transfer to 50ml Falcon tube.

Add 6ml of 1:2:1 solution (precipitation at RT)

1 volume of 2M Tris-HCl pH 8.2
2 volumes of 0.5M EDTA pH 8.0 (DEPC treated)
1 volume of 20% SDS

Add 3ml of phenol saturated with 0.1M Tris-HCl pH 8.2. Stir for minimum 15min.

Add 3ml chloroform. Stir for minimum 10min.

Centrifuge 4500rpm 15min.

Treat upper phase (aqueous) twice with 3ml chloroform, centrifuging 4500rpm 15min.

Transfer 4.5ml of aqueous phase to 30ml disposable polypropylene tube and add equal volume of ice-cold 6M LiCl. Precipitate O/N on ice at 4°C.

Day 2

Centrifuge 9000rpm 20min 4°C.

Resuspend into 1ml H2O, add 0.1ml 3M NaAcetate pH 5.2 and 3 volumes (3.3ml) of EtOH. Precipitate at RT for ca 60min.

Centrifuge 9000rpm 20min 16°C, rinse with 2ml 70% EtOH, centrifuge 9000rpm 5min, discard the supernatant, centrifuge again 9000rpm 2min, discard the supernatant, air dry pellet 10min and resuspend in 100 µl H2O.

Read OD260/280 with 2µl in 400µl H2O (nanodrop).

RNA Purification

Purify the RNA using RNeasy Mini Kit  from Qiagen n°74104

Dilute 150µg RNA  in a total volume of 100µl H2O

Add 350µl Buffer RLT + 3.5µl ß-mercaptoethanol

Add 250µl 100% EthOH

Transfer to the column

Centrifuge 15 sec at 12000 rpm and discard the flow-through

Transfer the column in a new collection tube

Add 500µl RPE buffer

Centrifuge 15 sec 12000 rpm and discard the flow-through

Centrifuge 1 min at 14000 rpm

Transfer the column in a new 1.5ml eppendorf tube and air dry 5 min

Elute the RNA in 2x 40µl H2O preheated at 50°C

Microcon

Transfer your 80µl RNA in a microcon column (NANOSEP 30K OMEGA from Life Sciences)

Centrifuge 3min at 9000g

Retrieve between 10-20µl of RNA over the membrane in a new 1.5ml eppendorf tube

Dilute 0.5µl of RNA in 50µl Tris-HCl pH=8.0 10mM and quantify with nanodrop

Probe Preparation

Prepare two 200 µl PCR tubes according to the recipe below:

100 µg of total RNA (Rneasy Cleanup is recommended¹)

2 µl oligo dT21mer (1 mg/ml)

enough water to achieve 13.4 ml final volume

1)             Heat 5 min at 70^oC in PCR machine, Leave at RT for 5 min

2)            Prepare mixes (one Cy3 and one Cy5) according to the following recipe

At RT: 

6 µl                  5x SuperScript II Buffer

3 µl                  0.1 M DTT

0.6 µl               dNTPS (25 mM dATP, dGTP, dTTP; 10 mM dCTP)

2 µl                  SuperScript II Reverse Transcriptase

2 µl                  RNase Inhibitor

divide 13.6 mL per tube (multiple tubes if necessary)

At RT:  

3 µl                  Cy3 (1 mM) for control

3 µl                  Cy5 (1 mM) for test

3)             Mix well and incubate at 42^oC for 2 hr in PCR machine

4)             Pool the two tubes together (corresponding Cy3 and Cy5)

5)             Add:     2.65 µl           25 mM EDTA

3.3 µl             1 M NaOH

6)             Incubate 10 min at 65^oC (PCR machine)

7)             Add:     3.3 µl             1 M HCl

5 µl                1 M Tris-HCl, pH 6.8

8)  Purify probe with Qiagen MinElute PCR purification kit (#28004)

1. Mix 5 volumes (370 µl) buffer PB with 1 vol of probe (74 µl)
2. Add 3 µl 3M NaAc. PH 5.2  and transfer to column
3. Spin 1 min at ? 10,000 x g.  Discard FT.
4. Add 700 µl buffer PE to column.
5. Spin 1 min at ? 10,000 x g.  Discard FT. Spin 1 additional min
6. Transfer column to new 1.5 mL tube, air dry 5min
7. Add 10 µl of buffer EB to the center of the membrane.  Wait 1 min then centrifuge for 1 min. Repeat the elution step  : ~20 µl eluate
8. Quantify using Nanodrop (2 µl/20). cDNA > 4 µg and dyes Cy3 and Cy5 > 30 pmoles each is OK

9)        Prepare Hybridization Solution according to volume and consistency needed:

Glass Chips (size depends on surface of DNA spots on the microarray):

80µl¹                         [Conc]_final

18                                                           labeled probe

39                                                           water

12                              3x                          20x SSC

8                                0.2                         tRNA (2mg/mL)

3                                0.38%                    10% SDS

¹25×60 lifter slips

Hybridization

1. Prepare slide in chamber with 10-13 ml 3x SSC per groove, with coverslip if using a lifter slip
2. Heat probe 1 min at 95^oC
3. Spin 1 min at high speed
4. Rapidly add probe to slide
5. Close chamber and immerge in 64^oC bath O/N.
6.  Washes

Washes

1. 5 min 2x SSC, 0.1% SDS
2. 5 min 2x SSC, 0.1% SDS
3. 1 min 0.2x SSC
4. 1 min 0.2x SSC
5. 1 min 0.1x SSC
6. 1 min 0.1x SSC
7. dry slide in centrifuge 2 min at 2600 rpm