| 1/3 | id: | 4 | | protocol_name: | Affy-Eukaryotic Target Preparation | | amount: | 10 | | label_used: | biotin | | amplification: | | | description: | SECTION 2 Eukaryotic Sample and Array Processing 2.1.10
Synthesis of Double-Stranded cDNA from Total RNA
This protocol is a supplement to instructions provided in the Invitrogen Life Technologies SuperScript Choice system. Please note the following before proceeding:
T7-oligo(dT) primer
5´ - GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24 - 3´
Starting material: High-quality total RNA (5.0 µg - 20.0 µg)
After purification, the RNA concentration is determined by absorbance at 260 nm on a spectrophotometer (one absorbance unit = 40 µg/mL RNA). The A260/A280 ratio should be approximately 2.0, with ranges between 1.9 to 2.1 considered acceptable. We recommend checking the quality of the RNA by running it on an agarose gel prior to starting the assay. The rRNA bands should be clear without any obvious smearing patterns from degradation. Before starting cDNA synthesis, the correct volumes of DEPC-treated H2O and Reverse Transcriptase (RT) must be determined. These volumes will depend on both the concentration and total volume of RNA that is being added to the reaction.
1. Users who do not purchase the GeneChip T7-Oligo(dT) Promoter Primer Kit may be required to obtain a license under U.S. Patent Nos. 5,569,584, 5,716,785, 5,891,636, 6,291,170, and 5,545,522 or to purchase another licensed kit.
For smaller amounts of starting material, please refer to the alternative protocol for
target labeling described in Small Sample Target Labeling Assay Version II, available
at www.affymetrix.com.
When using the GeneChip Sample Cleanup Module for the cDNA and IVT cRNA
cleanup steps, there is a potential risk of overloading the columns if greater than the
recommended amount of starting material is used.
Use Table 2.1.1 and Table 2.1.2 for variable component calculations. Determine the
volumes of RNA and SuperScript II RT required in Table 2.1.1, then calculate the
amount of DEPC-treated H2O needed in Step 1 Table 2.1.2 to bring the final First-
Strand Synthesis volume to 20 µL.
CHAPTER 1 Eukaryotic Target Preparation 2.1.11
Eukaryotic
Table 2.1.1
Reverse Transcriptase Volumes for First-Strand cDNA synthesis Reaction
Total RNA (ug) Superscript II room temp (µl), 200U/µl
5.0 to 8.0 1.0
8.1 to 16.0 2.0
16.1 to 20.0 3.0
The combined volume of RNA, DEPC-treated H2O and SuperScript II RT should not
exceed 11 µL as indicated in Table 2.1.2.
1. Primer Hybridization
T7-oligo (dT) primer, 50 µM 2 µl 5.0 to 20 µg
Incubate at 70oC for 10 minutes
Quick spin and put on ice RNA (variable)
2. Temperature Adjustment 5X First-strand cDNA buffer 4 µl
0.1 M DTT 2 µl
10 mM dNTP mix 1 µl
Add to the above tube and mix well
Incubate at 42oC for 2 minutes
3. First-strand synthesis Superscript II room temp (variable) see Table 2.1.1
Add to the above tube and mix well (200 U/µl)
Incubate at 42oC for 1 hour
The above incubations have been changed from the SuperScript protocols and are done at 42°C.
SECTION 2 Eukaryotic Sample and Array Processing 2.1.12
1. Place First-Strand reactions on ice. Centrifuge briefly to bring down condensation on sides of tube.
2. Add to the First-Strand synthesis tube the reagents listed in the following Second-
Strand Final Reaction Composition
Component Volume Final Concentrationm
DEPC-treated water 91 µl
5X Second-strand Reaction Buffer 30 µl 1X
10 mM dNTP 3 µl 200 uM,each
10 U/ul E. coli DNA ligase 1 µl 10 U
10 U/ul E. coli DNA Polymerase 4 µl 40 U
2 U/ul E.coli RNAse H 1µl 2 U
Final Volume 150 µl
3. Gently tap tube to mix. Then, briefly spin in a microcentrifuge to remove condensation and incubate at 16°C for 2 hours in a cooling waterbath.
4. Add 2 µL [10 U] T4 DNA Polymerase.
5. Return to 16°C for 5 minutes.
6. Add 10 µL 0.5M EDTA.
7. Proceed to cleanup procedure for cDNA, Cleanup of Double-Stranded cDNA on
page 2.1.15, or store at -20°C for later use.
CHAPTER 1 Eukaryotic Target Preparation 2.1.15
Eukaryotic
Cleanup of Double-Stranded cDNA
1. Add 600 µL cDNA Binding Buffer to the 162 µL final double-stranded cDNA synthesis preparation (page 2.1.10 or 2.1.13). Mix by vortexing for 3 seconds.
2. Check that the color of the mixture is yellow (similar to cDNA Binding Buffer without the cDNA synthesis reaction).
3. Apply 500 µL of the sample to the cDNA Cleanup Spin Column sitting in a 2 mL
Collection Tube, and centrifuge for 1 minute at >= 8,000 x g (>= 10,000 rpm).
Discard flow-through.
4. Reload the spin column with the remaining mixture (262 µL) and centrifuge as above. Discard flow-through and Collection Tube.
5. Transfer spin column into a new 2 mL Collection Tube (supplied). Pipet 750 µL cDNA Wash Buffer onto the spin column. Centrifuge for 1 minute at >= 8,000 x g
(>= 10,000 rpm). Discard flow-through.
6. Open the cap of the spin column and centrifuge for 5 minutes at maximum speed
(<= 25,000 x g). Discard flow-through and Collection Tube. Place columns into the centrifuge using every second bucket. Position caps over the adjoining bucket so that they are oriented in the opposite direction to the rotation (i.e., if the microcentrifuge rotates in a clockwise direction, orient the caps in a counterclockwise direction). This avoids damage of the caps. Centrifugation with open caps allows complete drying of the membrane.
BEFORE STARTING, please note:
_ cDNA Wash Buffer is supplied as a concentrate. Before using for the first time, add
24 mL of ethanol (96-100%), as indicated on the bottle, to obtain a working solution,
and checkmark the box on the left-hand side of the bottle label to avoid confusion.
_ All steps of the protocol should be performed at room temperature. During the
procedure, work without interruption.
_ If cDNA synthesis was performed in a reaction tube smaller than 1.5 mL, transfer the reaction mixture into a 1.5 or 2 mL microfuge tube (not supplied) prior to addition of cDNA Binding Buffer. If the color of the mixture is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow. cDNA Wash Buffer is supplied as a concentrate. Ensure that ethanol is added to the cDNA Wash Buffer before use (see IMPORTANT note above before starting).
7. Transfer spin column into a 1.5 mL Collection Tube, and pipet 14 µL of cDNA Elution Buffer directly onto the spin column membrane. Incubate for 1 minute at room
temperature and centrifuge 1 minute at maximum speed (<= 25,000 x g) to elute.
Ensure that the cDNA Elution Buffer is dispensed directly onto the membrane. The
average volume of eluate is 12 µL from 14 µL Elution Buffer.
8. An aliquot of the cDNA prepared from isolated poly-A RNA can be analyzed for size distribution and yield on a 1% agarose gel. One microliter of double-stranded cDNA should be sufficient to detect on an agarose gel stained with ethidium bromide. A representative gel is shown in cáÖìêÉ OKNKN on page 2.1.22. We do not recommend gel analysis for cDNA prepared from total RNA.
9. After cleanup, please proceed to Synthesis of Biotin-Labeled cRNA on page 2.1.17.
We do not recommend RNase treatment of the cDNA prior to the in vitro
transcription and labeling reaction; the carry-over ribosomal RNA does not seem to
inhibit the reaction. Quantifying the amount of double-stranded cDNA by absorbance at 260 nm is not recommended. The primer can contribute significantly to the absorbance. Subtracting the theoretical contribution of the primer based on the amount added to the reaction is not practical.
CHAPTER 1 Eukaryotic Target Preparation 2.1.17
Eukaryotic
Synthesis of Biotin-Labeled cRNA
1. Enzo® BioArray™ HighYield™ RNA Transcript Labeling Kit3 (Affymetrix, P/N
900182) is used for generating labeled cRNA target. Use the following tables to
determine the amount of cDNA used for each IVT reaction. Done properly, each
reaction should produce sufficient biotin-labeled target to hybridize to at least two
Standard Format (49 Format) GeneChip expression probe arrays in parallel.
The purity and quality of template cDNA is important for high yields of biotin-labeled
RNA. Use only RNase-free water, buffers, and pipette tips. Store all reagents at -20°C, in a freezer that is not self-defrosting. Prior to use, centrifuge all reagents briefly to ensure that the components remain at the bottom of the tube. The product should be used only until the expiration date stated on the label.
2. Add to RNase-free microfuge tubes template cDNA and additions of other reaction
components in the order indicated in the following table. Keep reactions at room
temperature while additions are made to avoid precipitation of DTT.
3. Carefully mix the reagents and collect the mixture in the bottom of the tube by brief (5 second) microcentrifugation.
4. Immediately place the tube in a 37°C water bath. Incubate for 4 to 5 hours, gently
mixing the contents of the tube every 30-45 minutes during the incubation.
5. Store labeled cRNA at -20°C, or -70°C (long-term storage) if not purifying
immediately.
6. Proceed to cRNA cleanup procedure, Cleanup and Quantification of Biotin-Labeled
cRNA on page 2.1.19 Each GeneChip® Sample Cleanup Module contains 30 cDNA cleanup columns and 30 IVT cRNA cleanup columns. If more than one IVT is carried out from a single cDNA sample and is purified on separate IVT cRNA cleanup columns, there will not be sufficient IVT cRNA columns in each kit for 30 samples.
Overnight incubation may produce shorter products, which is less desirable.
CHAPTER 1 Eukaryotic Target Preparation 2.1.19
Eukaryotic
Cleanup and Quantification of Biotin-Labeled cRNA
BEFORE STARTING please note:
_ It is essential to remove unincorporated NTPs, so that the concentration and purity of cRNA can be accurately determined by 260 nm absorbance.
_ DO NOT extract biotin-labeled RNA with phenol-chloroform. The biotin will cause some of the RNA to partition into the organic phase. This will result in low yields.
_ Save an aliquot of the unpurified IVT product for analysis by gel electrophoresis.
_ IVT cRNA Wash Buffer is supplied as a concentrate. Before using for the first time, add 20 mL of ethanol (96-100%) as indicated on the bottle to obtain a working solution, and checkmark the box on the left-hand side of the bottle label to avoid confusion.
_ IVT cRNA Binding Buffer may form a precipitate upon storage. If necessary, redissolve by warming in a water bath at 30°C, and then place the buffer at room temperature.
_ All steps of the protocol should be performed at room temperature. During the
procedure, work without interruption. IVT cRNA Wash Buffer is supplied as a concentrate. Ensure that ethanol is added to the IVT cRNA Wash Buffer before use (see IMPORTANT note above before starting).
1. Add 60 µL of RNase-free water to the in vitro transcription reaction and mix by
vortexing for 3 seconds.
2. Add 350 µL IVT cRNA Binding Buffer to the sample and mix by vortexing for
3 seconds.
3. Add 250 µL ethanol (96-100%) to the lysate, and mix well by pipetting. Do not
centrifuge.
4. Apply sample (700 µL) to the IVT cRNA Cleanup Spin Column sitting in a 2 mL
Collection Tube. Centrifuge for 15 seconds at >= 8,000 x g (>= 10,000 rpm). Discard flow-through and Collection Tube.
5. Transfer the spin column into a new 2 mL Collection Tube (supplied). Pipet 500 µL
IVT cRNA Wash Buffer onto the spin column. Centrifuge for 15 seconds at >= 8,000 x g (>= 10,000 rpm) to wash. Discard flow-through. 6. Pipet 500 µL 80% (v/v) ethanol onto the spin column and centrifuge for 15 seconds at >= 8,000 x g (>= 10,000 rpm). Discard flow-through.
7. Open the cap of the spin column and centrifuge for 5 minutes at maximum speed
(<= 25,000 x g). Discard flow-through and Collection Tube. Place columns into the centrifuge using every second bucket. Position caps over the adjoining bucket so that they are oriented in the opposite direction to the rotation (i.e., if the microcentrifuge rotates in a clockwise direction, orient the caps in a counterclockwise direction). This avoids damage of the caps. Centrifugation with open caps allows complete drying of the membrane.
8. Transfer spin column into a new 1.5 mL Collection Tube (supplied), and pipet 11 µL of RNase-free water directly onto the spin column membrane. Ensure that the water is dispensed directly onto the membrane. Centrifuge 1 minute at maximum speed (<= 25,000 x g) to elute.
9. Pipet 10 µL of RNase-free water directly onto the spin column membrane. Ensure that the water is dispensed directly onto the membrane. Centrifuge 1 minute at maximum speed (<= 25,000 x g) to elute.
For subsequent photometric quantification of the purified cRNA, we recommend
dilution of the eluate between 1:100 fold and 1:200 fold.
Use spectrophotometric analysis to determine the cRNA yield. Apply the convention that 1 absorbance unit at 260 nm equals 40 µg/mL RNA.
For quantification of cRNA when using total RNA as starting material, an adjusted cRNA yield must be calculated to reflect carryover of unlabeled total RNA. Using an estimate of 100% carryover, use the formula below to determine adjusted cRNA yield:
adjusted cRNA yield = RNAm - (total RNAi)(y)
RNAm = amount of cRNA measured after IVT (µg)
total RNAi = starting amount of total RNA (µg)
y = fraction of cDNA reaction used in IVT
Example: Starting with 10 µg total RNA, 50% of the cDNA reaction is added to the IVT,
giving a yield of 50 µg cRNA. Therefore, adjusted cRNA yield = 50 µg cRNA - (10 µg total RNA) (0.5 cDNA reaction) = 45.0 µg.
Use adjusted yield in Eukaryotic Target Hybridization on page 2.3.3.
The minimum concentration for purified cRNA is 0.6 µg/µL before starting the
following fragmentation reaction in "Fragmenting the cRNA for Target
Preparation" on page 2.1.21.
Please refer to Table 2.3.1 on page 2.3.7 for the amount of cRNA required for one
array hybridization experiment. The amount varies depending on the array format.
Please refer to the specific probe array package insert for information on the array
format.
CHAPTER 1 Eukaryotic Target Preparation 2.1.21
Eukaryotic
Gel electrophoresis of the IVT product is done to estimate the yield and size distribution of labeled transcripts. Parallel gel runs of unpurified and purified IVT product can help determine the extent of a loss of sample during the cleanup process.
Fragmenting the cRNA
for Target Preparation
Fragmentation of cRNA target before hybridization onto GeneChip probe arrays has been shown to be critical in obtaining optimal assay sensitivity.
Affymetrix recommends that the cRNA used in the fragmentation procedure be sufficiently concentrated to maintain a small volume during the procedure. This will minimize the amount of magnesium in the final hybridization cocktail. The cRNA must be at a minimum concentration of 0.6 µg/µL. Fragment an appropriate amount of cRNA for hybridization cocktail and gel analysis (see Section 2, Chapter 3, Table 2.3.1).
1. Add 2 µL of 5X Fragmentation Buffer for every 8 µL of RNA plus H2O. The
fragmentation buffer has been optimized to break down full-length cRNA to 35 to 200 base fragments by metal-induced hydrolysis.
The final concentration of RNA in the fragmentation mix can range from 0.5 µg/µL to
2 µg/µL. The following table shows an example of a fragmentation mix for a 20 µg
cRNA sample at a final concentration of 0.5 µg/µL.
For fragmentation, use adjusted cRNA concentration, as described in Step 2:
Quantification of the cRNA on page 2.1.20.
Example for 0.5 µg/µL final concentration:
2. Incubate at 94°C for 35 minutes. Put on ice following the incubation.
3. Save an aliquot for gel analysis.
At least 1 µg fragmented cRNA is needed if using ethidium bromide for staining the
gel. Less RNA can be used with SYBR Green II staining. See Step 3: Checking
Unfragmented Samples by Gel Electrophoresis on page 2.1.21, for information
regarding gel electrophoresis. The standard fragmentation procedure should produce a distribution of RNA fragment sizes from approximately 35 to 200 bases. An example of a gel with cRNA samples before and after fragmentation is shown below.
4. Store undiluted, fragmented sample RNA at -20°C until ready to perform the
hybridization, | | remark: | | | last_update: | 2003-09-16 22:38:56 | | login_id: | caldo | | resource: | Affymetrix manual | | amount_unit: | ug |
| 2/3 | id: | 5 | | protocol_name: | Amino Allyl MessageAmp2 aRNA Amplification Kit | | amount: | 0 | | label_used: | | | amplification: | T7 RNA Polymerase | | description: | Every step is identical to manufacture\\\'s protocol | | remark: | | | last_update: | 2005-10-05 19:51:05 | | login_id: | HakeOligo | | resource: | | | amount_unit: | ug |
| 3/3 | id: | 6 | | protocol_name: | Indirect Labeling with Cy3 and Cy 5 | | amount: | 50 | | label_used: | | | amplification: | none | | description: | indirect labelling using aminoallyl-dUTP | | remark: | | | last_update: | 2005-10-07 16:04:27 | | login_id: | HakeOligo | | resource: | other | | amount_unit: | ug |
|