GeneChip Eukaryotic Small Sample Target Labeling

Step 1

The sample RNA and T&-(dT)24 primer were mixed in a microcentrifuge tube

	Volume/Reaction	Final Concentration
DEPC-treated water	9 µl
Total RNA, variable concentration	1 µl	variable, <100 ng
T7-(dT)24 primer, 100 pmol/µl	1 µl	100 pmol/reaction
Total Volume	11 µl

The RNA/primer mix was denatured by incubation at 70°C for 10 minutes
Reaction mix was cooled on ice for 2 minutes

The following reagents were added to the above RNA/primer mixture for the first strand cDNA synthesis:

	Volume/Reaction	Final Concentration
RNA/primer mixture	11 µl
5X first strand buffer	4 µl	1X
DTT, 0.1 mM	2 µl	10 mM
dNTP mix, 10 mM	1 µl	500 µM
RNase inhibitor, 40 U/µl	1 µl	40 U/reaction
Total Volume	19 µl

The reaction was incubated at 42°C for 2 minutes
One µl SuperScript II (200 U/µl) was added to the above reaction to make a final volume of 20 µl
The reagents were mixed gently and spun in a microcentrifuge briefly before incubating at 42°C for 1 hour

Step 2: First Cycle, Second Strand cDNA Synthesis

The first strand reaction was spun down and cooled on ice

	Volume/Reaction	Final Concentration
First strand synthesis reaction	20 µl
DEPC-treated water	91 µl
5X second strand buffer	30 µl	1X
dNTP mix, 10 mM	3 µl	200 µM
DNA ligase, E.coli, 10 U/µl	1 µl	10 U/reaction
DNA polymerase, E. coli,10 U/µl	4 µl	40 U/reaction
RNase H, 2 U/µl	1 µl	2 U/reaction
Total Volume	150 µl

The reaction was mixed gently and spun in a microcentrifuge briefly followed by incubation at 16°C for 2 hours
To fill in the ends of the double-stranded cDNA, 2 µl of T4 DNA polymerase (5 U/µl) was added to the above reaction and incubated at 16°C for an additional 15 minutes

Step 3: First Cycle, Double-Stranded cDNA Cleanup by Ethanol Precipitation

To the reaction tube, 1µl of 5 mg/ml glycogen, 0.6 volume of 5M NH₄OAc (92 µl) and 2.5 volumes of cold absolute ethanol (612 µl) were added
The content was mixed thoroughly and centrifuged immediately at 14,000 rpm for 20 minutes at 4°C
The pellet was washed by adding 1 ml of 70% cold ethanol and centrifuged at 14,000 rpm for 5 minutes
After removing the ethanol carefully, a speed vacuum was used for approximately 5-10 minutes to dry the pellet. Avoid over-drying of the DNA. The pellet was stored at 4°C or 20°C overnight before proceeding to the IVT reaction

Step 4: First Cycle, IVT for cRNA Amplification Using Ambion MEGAscript T7 Kit

The following reagents were added to the above dried double-stranded cDNA pellet at room temperature in the Ambion MEGAscript protocol:

	Volume/Reaction	Final Concentration
DEPC-treated water	8 µl
ATP, 75 mM	2 µl	7.5 mM
GTP, 75 mM	2 µl	7.5 mM
CTP, 75 mM	2 µl	7.5 mM
UTP, 75 mM	2 µl	7.5 mM
10X reaction buffer	2 µl	1X
Ambion Enzyme mix	2 µl
Total Volume	20 µl

The reaction was mixed gently and spun in the microcentrifuge tube briefly before incubating at 37°C for four hours.

Step 5: First Cycle, cRNA Cleanup with RNeasy Columns

Eighty microliters of RNase-free water was added to the above cRNA product
The RNeasy Mini column was used for cRNA purification following the protocol for RNA Cleanup handbook from Qiagen that accompanies the RNeasy Mini kit
In the last step of cRNA purification, the product was eluted with 50 µl of RNase-free water once
The cRNA yield was determined by measuring the A₂₆₀. Due to the limited amount of cRNA the measurement may not be accurate. However, the rough estimation may help to determine the amount of cRNA to be used in the second cycle. The cRNA could be stored at -20°C at this point before proceeding to the next step.

Second Cycle of Amplification

Step 6: Second Cycle, First Strand cDNA Synthesis

The following table was used as a general guideline to determine the amount of cRNA to be used in the second cycle:

Total Starting Material	cRNA To Be Used for Second Amplification
50-100 ng	100-250 ng
<20 ng	all 50 µl

Note: For instances when the yield was less than that indicated in the table, all of the elution (50 µl) was used in the second cycle of cDNA synthesis. For the reaction where the volume of cRNA needed 10 µl, a speed vacuum was used to reduce the volume to 10 µl before proceeding to the next step.

The cRNA and random primers were mixed in a microcentrifuge tube:

	Volume/Reaction	Final Concentration
cRNA variable	up to 10 µl	variable
Random primers, 1 µg/µl	1 µl	1 µg/reaction
DEPC-treated water	added to final volume of 11 µl
Total volume	11 µl

The RNA was denatured by incubation at 70°C for 10 minutes
The cRNA/primer mix was cooled on ice for 2 minutes

The following reagents were added to the above cRNA/primer mixture for the first strand cDNA synthesis:

	Volume/Reaction	Final Concentration
cRNA/primer mixture	11 µl
5X first strand buffer	4 µl	1X
DTT, 0.1 M	2 µl	10 mM
dNTP mix, 10 mM	1 µl	500 µl
RNase inhibitor, 40 U/µl	1 µl	40 U/reaction
Total Volume	19 µl

The reaction was incubated at 42°C for 2 minutes
One microliter of SuperScript II (200 U/µl) was added to the above reaction to make a final volume of 20 µl
The reagents were mixed gently and spun down in the microcentrifuge briefly before incubating at 42°C for 1 hour
One microliter of RNase H (2 U/µl) was added and incubated for 20 minutes at 37°C
The reaction was heated to 95°C for 5 minutes to denature the RNase H and separate the DNA/RNA hybrids
The reaction was then chilled on ice

Step 7: Second Cycle, Second Strand cDNA Synthesis with T7-(dT)24 Primer

The first strand reaction was spun down in a microcentrifuge, then 1 µl of 100 pmol/µl T7-(dT)24 primer was added
The mix was incubated at 70°C for 10 minutes and then cooled on ice
The following reagents were added to the reaction tube for the second strand cDNA synthesis:

	Volume/Reaction	Final Concentration
First strand mix/T7-(dT)24 primer	22 µl
DEPC-treated water	91 µl
5X second strand buffer	30 µl	1X
dNTP mix, 10 mM	3 µl	200 µM
DNA polymerase, E. coli, 10 U/µl	4 µl	40 U/reaction
Total Volume	150 µl

The reaction was mixed gently and spun in the microcentrifuge tube briefly before incubating at 16°C for 2 hours
To fill the ends of the double-stranded cDNA, 2 µl of T4 DNA polymerase (5 U/µl) was added to the above reaction and the reaction was incubated at 16°C for an additional 15 minutes

Step 8: Second Cycle, Double-Stranded cDNA Cleanup by Ethanol Precipitation

To the reaction tube, 1µl of 5 mg/ml glycogen, 0.6 volume of 5M NH₄OAc (92 µl) and 2.5 volumes of cold absolute ethanol (612 µl) were added
The content was mixed thoroughly and centrifuged immediately at 14,000 rpm for 20 minutes at 4°C
The pellet was washed by adding 1 ml of 70% cold ethanol and centrifuged at 14,000 rpm for 20 minutes at 4°C
Ethanol was removed carefully and put in a speed vacuum for approximately 5-10 minutes to dry the pellet stored at 4°C or -20°C overnight before proceeding to the IVT reaction

Step 9: Second Cycle, IVT for cRNA Amplification & Labeling with ENZO Bioarray HighYield RNA Kit

The following reagents were added to the above dried double-stranded cDNA pellet at room temperature in the ENZO kit product insert:

	Volume/Reaction	Final Concentration
DEPC-treated water	22 µl
10X HY reaction buffer	4 µl	1X
10X Biotin-labeled ribonucleotides	4 µl	1X
10X DTT	4 µl	1X
10X RNase inhibitor mix	4 µl	1X
20X T7 RNA polymerase	2 µl	1X
Total Volume	40 µl

The reaction was mixed gently and spun in the microcentrifuge tube briefly before incubating at 37°C for 4 hours.

Step 10: Second Cycle, Labeled cRNA Target Cleanup with RNeasy Columns

Sixty microliters of RNase-free water was added to the above cRNA product
The RNeasy mini column was used for cRNA purification. Protocol for RNA Cleanup handbook from Qiagen RNeasy Mini Kit was followed
In the last step of cRNA purification, the product was eluted with 50 µl of RNase-free water once
Two microliters of the labeled cRNA was removed and added to 98 µl of water to measure the absorbance at 260 nm for determination of the cRNA yield
Ten micrograms of labeled cRNA was then fragmented and hybridized to GeneChip probe arrays as described in the GeneChip Expression Analysis Technical manual