Sample processing protocol for GG-H array


Generation of Amplified cRNA from Total RNA to be performed with the WT Expression Kit (Ambion, Inc./Applied Biosystems, cat# 4411974)



1. First strand cDNA synthesis

a. Bring 50 ng of total RNA to 5 ml Nuclease-Free Water
b. Perform the next step at room temperature; doing so on ice will cause a significant drop in yield.
c. Prepare Ambion Wt Expression first strand master mix as follows:

  First strand buffer mix 4 mL
  First strand enzyme mix 1 mL
  Total volume 5 mL

d. Add 5 mL of the first strand master mix to a supplied 0.5 mL PCR tube
e. Add 5 ml of total RNA to the same tube
f. Mix well by pipetting or gentle vortexing
f.  Incubate for 1 hour at 25˚, then for 1 hour at 42 ˚, then for 2 minutes at 4˚
Note: cover of thermal cycler must be set at 50˚ or sample quality will be compromised. An ABI Veriti 96-well thermal cycler is preferred.
g. After the reaction is done, place samples on ice for at least 2 minutes, then immediately proceed to the next step.

 

2. Second strand cDNA synthesis:

a. On ice, prepare the Ambion Wt Expression second strand master mix as follows:

  Nuclease-Free water 32.5 mL
  Second strand buffer mix 12.5 mL
  Second strand enzyme mix 5 mL
  Total Volume 50 mL

b. Add 50 mL of the second strand master mix to each 10 mL reaction. Mix well by flicking the tubes, then spin briefly
c. Incubate for 1 hr at 16˚, the for 10 minutes at 65˚, then for 2 minutes at 4˚
Note: the cover of the thermal cycler must be disabled or left completely off during the reaction
d. After the reaction has finished, place the tubes on ice and proceed to the next step.


3. In Vitro Transcription

a. At room temperature, prepare the Ambion Wt Expression IVT master mix as follows:

  IVT buffer mix 24 mL
  IVT enzyme mix 6 mL
  Total volume 30 mL

b. Add 30 mL of the IVT master mix to each 60 uL reaction, mix by gently vortexing, then spin briefly
c. Incubate for 16 hr at 40˚, then at 4˚
Note: cover of thermal cycler must be set at 50˚ or sample quality will be compromised
d. Place the tubes on ice and proceed to the reaction cleanup


4. Cleanup of cRNA (using Ambion Wt Expression reagents)

Note:  Before the purification is started, it is necessary to do the following:

a. At room temperature, prepare cRNA binding mix as follows:

  Nucleic acid binding beads       10 mL
  Nucleic acid binding buffer concentrate 50 mL
  Total volume 60 mL

b. Add 60 mL cRNA binding mix to each sample and pipet up and down 3 times to mix
c. Transfer sample to a well of the provided U-bottom plate
d. Add 60 mL 100% isopropanol to each sample and pipet up and down 3 times to mix.
Note: do not use ethanol at this step
e. Shake plate gently (40% maximum speed) for 2 or more minutes to bind cRNA to the beads.
f. Place the plate on the magnet and allow to sit for ~5 minutes (or until the mixture is transparent).
g. Aspirate and discard the supernatant without disturbing the beads, then take the plate off the magnetic stand.
h. Add 100 mL Nucleic acid wash solution to each sample and shake the plate for 1 minute at moderate speed (70% maximum speed setting on shaker)
i. Place the plate on the magnetic stand and allow to sit for 1 minute or until the solution is transparent
j. Aspirate and discard the supernatant without disturbing the beads and take plate off magnet
k. Repeat steps h-j to wash the sample one more time
l. Shake the plate at maximum speed for 1 minute to dry the beads until no more liquid is visible without over drying the beads.
m. Add 40 mL preheated elution solution to the beads and incubate at room temperature without shaking for 2 minutes
n. Shake the plate at maximum speed for 3 minutes to make sure the beads are fully dispersed. If not, additional pipetting may be necessary.
o. Place the plate on the magnet and leave it until the solutions becomes transparent.
p. Transfer the supernatant to a 1.5 mL Eppendorf tube and check concentration on NanoDrop™ spectrophotometer.
Generation of second cycle cDNA to be performed with reagents purchased individually.


5. Second cycle, first strand cDNA synthesis

a. Take 15 mg of cRNA from previous step and split into three 5 mg reactions. Bring the volume up to 7 mL with nuclease-free water
b. Prepare the primer annealing master mix as follows:

  cRNA (5 mg)       7 mL
  Random primers 3 mg/ml (Invitrogen, cat#48190-011) 1 mL
  Total volume 8 mL

c. Mix well by flicking the tubes.
d. Incubate for 5 minutes at 70˚, then 5 minutes at 25˚ and then cool to 4˚ for at least 2 minutes and place on ice
e. Prepare the first strand master mix as follows:

  5X first strand buffer (included with SSII) 4 mL
  DTT, 0.1 M (included with SSII)  2 mL
  dNTP mix, 10 mM (4dTTP:1dUTP, Invitrogen cat# U1330, U119)* 1 mL
  RNaseOUT, 40 U/mL (Invitrogen, cat# 10777022)  1 mL
  Superscript II, 200 U/mL (Invitrogen, cat# 18064014) 4 mL
  Total Volume 12 mL

*Mix 5 μL of 100 mM dATP, 5 μL of 100 mM dCTP , 5 μL of 100 mM dGTP, 4 μL of 100 mM dTTP, and 1 μL of 100 mM dUTP with 30 μL of  RNase-free Water.

f. Add 12 mL first strand master mix to each tube and mix by flicking the tubes and spin briefly
g. Incubate for 5 minutes at 25˚, then for 1 hr at 42˚, then 4˚ for at least 2 minutes.
h. When the reaction has finished, place the tubes on ice.

 

6. Second cycle second strand synthesis

a. Prepare the second strand master mix as follows:

  1M MgCl2, diluted to 17.5 mM (Ambion, cat# 9530G) 8 mL
  dNTP, 10 mM (4dTTP:1dUTP, Invitrogen cat# U1330, U119) 0.6 mL
  Klenow 3’→5’ exo-, 5 U/uL (NEB cat# M0212L) 5.4 mL
  RNase-free water 5.5 mL
  RNaseH, 2 U/uL (NEB cat# M0297L) 0.5 mL
  Total Volume 20 mL

b. Add 20 mL of the second strand master mix to each tube for a total of 40 mL. Mix by flicking the tubes and then spin briefly
c. Incubate the samples for 40 minutes at 37˚, then for 10 minutes at 75˚, then 4˚ for at least 2 minutes. Then proceed to cleanup


7. Cleanup of second cycle cDNA

Note: this protocol requires the use of the Affymetrix GeneChip™ sample cleanup module (Affymetrix, cat# 900371). Be sure that 100% ethanol has been added to the wash buffer
a. Add 60 mL Rnase-free water to each sample and add 370 mL cDNA binding buffer and vortex for 3 seconds.
b. Add 470 mL to a cDNA cleanup column and spin at ≥8,000 x g for 1 minute. Discard flow-through and collection tube
c. Place spin column in a new 2 mL collection tube and add 750 mL cDNA wash buffer. Spin at ≥8,000 x g for 1 minute and discard flow-through
d. Open the cap of the column and spin at maximum speed for 5 minutes. Discard flow-through and place column in a 1.5 mL Eppendorf tube
e. Add 18 mL of cDNA elution buffer to the column and incubate at room temperature for 1 minute
f. Spin at full speed for 1 minute
g. Repeat steps e and f to elute one more time, then combine the three reactions back into one and measure concentration on Nanodrop™ spectrophotometer.


8. Fragmentation of cDNA

a. Take 20 mg cDNA and split into three separate reactions.
b. Prepare the fragmentation reaction as follows:

  cDNA (6.67 mg) 32.2 mL
  NEB buffer 4, 10X 4.8 mL
  Uracil DNA Glycosylase 2 U/mL (NEB cat# M0280S) 4 mL
  APE 1 10 U/uL (NEB cat#M0282S) 7 mL
  Total Volume 48 mL

c. Flick the tubes and spin briefly
d. Incubate reactions for 1 hour at 37˚, then for 1 minute at 93˚, then at 4˚ for at least 2 minutes
e. Place the samples on ice and proceed to the next step
Note: 3 uL can be saved here for Agilent Bioanalyzer analysis

 

9. Labeling of fragmented cDNA

Note: this step requires the use of the Affymetrix GeneChip™ WT Terminal Labeling Kit (Affymetrix, cat# 900671)

a. Prepare the labeling reaction as follows:

  Fragmented cDNA  45 mL
  5X TdT reaction buffer 12 mL
  rTdT, (30 U/mL)  2 mL
  DLR-1a, 5 mM 1 mL
  Total Volume 60 mL

b. Flick the tubes to mix and spin briefly
c. Incubate the reactions for 60 minutes at 37˚ and then at 4˚ for at least 2 minutes
d. Add 2 mL 0.5M EDTA to stop the reactions, flick to mix and spin briefly

 

10. Concentration of three samples into one

a. Add all three reactions to one YM-3 Microcon column inserted into a 1.5 mL tube
b. Centrifuge at 14,000 x g for 40-50 minutes. At 30 minutes, start measuring the flow-through until the volume is close to 100 mL
c. In a new tube, place column upside down and spin at 1000 x g for 3 minutes. Try to have a little less than 68.7 mL and bring the volume up to 68.7 mL with water

 

11. Hybridization of fragmented, labeled cDNA to array

a. Prepare the hybridization mix in a 1.5mL eppendorf tube as follows:

  Fragmented, labeled cDNA 68.7 mL
  Control oligo B2, 3nM (Affymetrix, cat# 900364) 3.3 mL
  20X Eukaryotic hyb controls (Affymetrix, cat# 900458) 10 mL
  Herring sperm DNA, 10 mg/mL (Promega, cat# D1811) 2 mL
  Acetylated BSA, 50 mg/mL (Invitrogen, cat# 15561-020) 2 mL
  2X Hybridization Buffer* 100 mL
  DMSO (Sigma, cat# D5879) 14 mL
  Total volume 200 mL

            * In–house reagent preparation instructions following the protocol

b. Flick to mix and spin briefly
c. Heat the tubes for 5 minutes at 99˚, then for 5 minutes at 45˚
d. Add 200 mL 1X hybridization buffer* to each array and rotate in the hybridization oven at 45˚ for 10 minutes
e. Spin the tubes at full speed for 1 minute
f. After the arrays have finished the pre-hybridization, remove the 1X hybridization buffer
g. Add 200 mL of the sample to the array and hybridize in the oven for 16 hours at 45˚ rotating at 60 rpm.
h. After the hybridization is complete, remove the sample from the array and store at -20. Add 200 mL wash A*
* In–house reagent preparation instructions following the protocol

 

12. Washing, Staining and Scanning of the array

a. Follow the procedures in the Affymetrix GeneChip™ expression analysis technical manual to wash, stain and scan the arrays on the Affymetrix fluidics station 450 and the GeneChip™ Scanner 30007G. Use the wash protocol Euk-GE WS2.v4.

 


 

Appendix-Reagent Preparation

a. 2X hybridization buffer

  12X MES stock* 8.3 mL
  5M NaCl (Ambion, cat# AM9759) 17.7 mL
  0.5M EDTA (Ambion, cat# AM9260G) 4.0 mL
  10% Tween-20 (Pierce Biochemical, cat#28320) 0.1 mL
  Water   19.9 mL
  Total volume 50 mL

b. 12X MES stock

  MES free acid monohydrate (sigma, cat# M5287) 70.4 mL
  MES sodium salt (sigma, cat# M3885) 193.3 mL
  Molecular biology grade water 800 mL

Mix and adjust volume to 1000 mL. pH should be between 6.5 and 6.7. Filter through a 0.2 mM filter

c. Wash A

  20X SSPE (Invitrogen, cat#15591-043)  300 mL
  10% Tween-20 (Pierce Biochemical, cat#28320)  1.0 mL
  Water 698 mL

Filter through a 0.2 mM filter.

 


Last modified 12/13/2010