Expression profiling using genechip affymetrix technology

Steps:

1.     cDNA synthesis

2.     cDNA purification

3.     IVT Reaction

4.     cRNA fragmentation

5.     Target clean-up and hybridization

6.     Washing and staining arrays

A list of reagents can be found on the bottom of this document

 

PROCEDURE:

 

cDNA synthesis

For best results we recommend starting with 8ug of total RNA or 0.5ug of mRNA

 

1^st strand cDNA synthesis

Reagent	vol. ml
RNA + H2O	11
*T7T24 primer 100pm/ul	1

-       *Add primer DIRECTLY into sample

-       Incubate on heat block at 70°C for 10min., place on ice

 

Add the following to RNA mix:

Reagent	vol. ml
5X 1st strand buffer	4
0.1 M DTT	2
10 mM dntp	1
SuperScript II RT (200units/ul)	1

-       Make 20% more if preparing a master mix

-       Incubate at 42°C for 1hr

 

2^nd strand cDNA synthesis

On ice add:

Reagent	vol. ml
Nuclease Free Water	91
5X 2nd strand buffer	30
10mM dntp	3
E coli DNA pol I (10 units/ul)	4
E coli DNA ligase (10 units/ul)	1
E coli RNAse H (2 units/ul)	1

-       Make 20% more if preparing a master mix

-       Incubate at 16°C for 2hr (use microcooler)

 

Add:

Reagent	vol. ml
T4 DNA polymerase (5units/ul)	2

-       Incubate at 16 °C for 5min.

-       Add 10ml 0.5M EDTA

-       Proceed to cDNA clean-up or store at -20°C

(* the above reagents provided by Invitrogen)

 

Purify ds cDNA using QIAquick PCR purification kit (Qiagen)

 

-       Add 5 volumes of Buffer PB to 1 volume of cDNA mixture

-       Apply half of the sample to the QIAquick column and centrifuge at > 10,000 rpm for 45sec.

-       Discard flow-through, apply the other half of the sample to the column, and centrifuge at > 10,000 rpm for 45sec

-       Discard flow-through and place column back into the same tube

-       To wash, add 750ml Buffer PE to the column and centrifuge at > 10,000 rpm for 45sec

-       Discard flow-through, place QIAquick column in new collection tube, centrifuge at maximum speed for 1min

-       Place QIAquick column in a clean 1.5-ml microfuge tube

-       To elute DNA: add 30 ul Buffer EB (10 mM Tris-Cl, pH 8.5) to the center of the QIAquick column, let stand for 1 min. then centrifuge for 1 min at maximum speed. Repeat for a total 60ml elution.

-       Speed vacuum dry the samples and resuspend in 22 ml Nuclease Free H2O

-       You may freeze at –20°C or continue to IVT reaction

Synthesis of Biotin-Labeled cRNA (using ENZO BioArray High Yield RNA Transcript Labeling Kit) – IVT Reaction

 

vol. ul	Reagent
X	Fraction of ds cDNA corresponding to 5ug total RNA input
Y	Nuclease Free H2O
4	10X Hy reaction buffer
4	10X Biotin labeled ribonucleotides
4	10X DTT
4	10X Rnase inhibitor
2	T7 RNA polymerase
40 ul total

X + Y = 22 ul in volume

-       Incubate at 37°C for 4–6 hours while gently mixing the reaction every 30 minutes

-       Clean up the collected cRNA using the QIAGEN RNeasy mini kit (follow protocol in handbook)

-       Quantify the cRNA using Beckman DU530 Spectrophotometer

Fragmentation of cRNA for target preparation

-       Begin with 15mg of cRNA (volume not to exceed 16 ml – speed vac cRNA if necessary and

resuspend to 16ml)

-       Add 4ml RNA fragmentation buffer FOR A TOTAL VOLUME OF 20uL

-       Incubate at 94 °C for 15 min.

-       Quick-spin sample

-       Incubate at 94 °C for 15 min.

-       Quick-spin sample and add hybridization mix

-       Place on ice until ready to hybridize on chip

HYBRIDIZATION MIX:

Reagent	Mini Array (µl)	Standard Array(µl)	Final concentration
15 µg fragmented cRNA in 20ul			0.05 µg/µl
DEPC Tx H2O	21.3	104
2X MES Hybridization Buffer	50	150	1X
Promega Herring Sperm DNA(10mg/ml)		3	0.1 mg/ml
BSA (50 mg/ml)	1	3	0.5 mg/ml
Control Oligonucleotide B2 (3 nM)	1.67	5	50 Pm
20 X Eukaryotic Hybridization Controls (BioB,BioC,BioD and cre)	5	15	1.5, 5, 25, 100 pM respectively
Final volume	100 ul	300 ul

 

* Pre-warm 20 X controls and herring sperm DNA at 70 C for 5 min

* Pre-warm control oligo B2 and BSA at 37 C for 5 min

 

Target Cleanup and Hybridization

Chip Pre-treatment

-       Place the gene chip in a 45°C oven for 15 min.

-       Fill the chip with pre-warmed (45 °C) pretreatment solution

-       Place the chip back in the 45 °C oven and incubate for 15 min.

PRETREATMENT SOLUTION:

Reagent	Add (µl)	Final concentration
1X MES Hyb buffer	294
Ac-BSA (50 mg/ml)	3	0.5 mg/ml
Promega Herring Sperm DNA (10mg/ml)	3	0.1 mg/ml

 

Prepare for hybridization

-       Incubate hybridization mix at 99 °C for 5min. followed by another incubation at 45°C for 5 min.

-       Centrifuge hybridization mix for 10 min. at 14000rpm

-       Transfer mix to a fresh tube (avoid collecting the PPT - leave the last 10-20 µl)

-       Incubate hybridization mix at 45 °C for 5 min.

-       Remove pretreatment solution from chip

-       Fill the chip with hybridization mix

-       Cover septa with Tough Spots (microfuge stickers)

-       Incubate chips for 16-18hrs in gene chip hybridization oven set at 45°C (50°C for Human Chips)

 

Washing and Staining Probe Arrays

SAPE stain (600 µl) –wrap solution in FOIL

Reagent	Add (µl)	Final concentration
2X Stain Buffer	300	1X
DEPC Tx H2O	270
BSA (50 mg/ml)	24	0.5 mg/ml
SAPE (1mg/ml)	6	10 µg /ml

 

AB stain (300 µl) – wrap solution in FOIL

Reagent	Add (ul)	Final concentration
2X Stain Buffer	150	1X
DEPC Tx H2O	133.2
BSA (50 mg/ml)	12	0.5 mg/ml
Normal goat IgG in PBS (10 mg/ml)	3	0.1 mg/ml
Biotinylated antibody (500 ug/ml)	1.8	1.25 µg/ml

 

-       Pre-warm affymetrix buffer B (500µL per chip) at 45°C for 10 min

-       Remove sample from chip and store in 1.5mL tube at –20°C

-       Fill the chip with affymetrix buffer A (room temp)

-       Wash chips on GNF fluidics station

-       Remove 6X SSPE, add 200µL pre-warmed buffer B, and rotate 45°C for 5 min

-       Remove buffer B and repeat above step

-       Cool chips with buffer A

-       Remove buffer A, add SAPE,

-       Wash chip on GNF fluidics station

-       Remove 6X SSPE, add antibody (AB) stain, and incubate chips FACE DOWN and away from light at RT for 10min.

-       Remove stain buffer and rinse with 200µL buffer A

-       Stain with SAPE at room temp for 10 min, FACE DOWN

-       Wash the chip on GNF fluidics station

-       Remove 6X SSPE, carefully fill chip with 1X MES and cover septa with Tough Spots

 

Reagents

12X MES stock (100 ml) 1.22 M MES pH should be 6.5-6.7 (without adjustment)

Reagent	Add (g)
MES free acid monohydrate	7.04
MES Sodium Salt	19.3

bring up to 100 ml DEPC water

0.2 µm filter sterilize and store at 4 °C

 

2X MES Hybridization Buffer (500 ml)

Reagent	Add (ml)	Final 2X concentration
DEPC water	216
5M NaCl	200	2 M
12X MES stock	82	200 mM

0.2 µm filter sterilize

store at room temperature for a few weeks or 4C several months

 

2X Stain Buffer (250 ml) (Final 1x concentration,: 100 mM MES, 1M Na+, 0.005% Tween 20)

Reagent	Add (ml)
12X MES stock	41.7
5M NaCl	92.5
10% Tween 20	2.5
DEPC water	112.8

0.2 µm filter sterilize

store at room temperature for a few weeks or 4C several months

 

6XSSPE-Washing buffer (5L) (pH should be ~7.5-7.6 without adjustment)

Reagent	Add (ml)	Final concentration
20X SSPE	1500	6X
MQ water­­	Add water to 5L

0.2 µm filter sterilize

add to the filtered solution

10% Triton X-100

5

0.01 %

 

 

Buffer B (Affymetrix): Stringent Wash Buffer (500ml)

Reagent	Add (ml)
12X MES stock	41.65
5 M NaCl	2.6
DEPC water	455.25
10% Tween 20	0.5

0.2 µm filter sterilize

 

Buffer A (Affymetrix): Non-Stringent Wash Buffer (500ml)

Reagent	Add (ml)
20X SSPE	150
water­­	349
10% Tween 20	0.5

0.2 µm filter sterilize

 

2X Stain Buffer (250ml)

Reagent	Add (ml)
12X MES stock	41.65
5 M NaCl	92.5
DEPC water	112.8
10% Tween 20