Prior to Labeling RNA- DNase treatment.
All Materials are included below (Labeling Step)
For each sample: Add 1ul of DNaseI (10U)/40ug of total bacterial RNA in 1X
Amersham 1 Phor-All-Buffer.
Incubate for 20 min @ 37C
Stop RXN by adding EDTA & place sample over a Qiagen RNeasy Mini column as
per manufacturer recommendations.
Elute with 45ul of Nuclease free H2O (not DEPC treated).
Typical yields are ~800 ng/ul & run on gel.
Labeling S. aureus RNA (Antisense GeneChip): Procedure taken from
Affymetrix Pseudomonas protocol, with minor changes.
Materials:
100 mM dNTPs (Amersham PN 27-203501)
Random Primers (Invitrogen PN 48190-011)
SuperScript II RT (Invitrogen PN 18064-071)
SUPERase (Ambion PN 2696)
Nuclease-free Water (Ambion PN 9930)
1N NaOH
1N HCl
QIAQuick PCR Purification Kit
10X One-Phor-All buffer (Amersham PN 27-0901-02)
DNase I (Amersham PN 27-0514-01)
Enzo BioArray Terminal Labeling Kit (Affymetrix 900181)
0.5 M EDTA pH 8.0
1. cDNA Synthesis- in a 0.5 ml thin-walled (PCR) Eppendorff tube prepare
following mixture (30 ul):
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X ul total bacterial RNA (10 ug total)
10 ul of 75 ng/ul Random primers
2 ul of “Spike-in” controls
18 ul – Xul of Nuclease-free water
Mix well by pipetting
2. Incubate for 10 min at 700C; 250C for 10 min; 40C in a Thermocycler.
3. Add cDNA Synthesis Mixture (30 ul) per RXN:
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12 ul 1st Strand Buffer
6 ul 100mM DTT
3 ul 10 mM dNTP mix
1.5 ul 20 U/ul SUPERase-In
7.5 ul 200U/ul SuperScript II
Note: This cDNA Synthesis Mix is 1X, scale up accordingly.
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Incubate for 10 min @ 250C; 60 min @ 370C; 60 min @ 420C.
Stop reaction by incubating 10 min @ 700C; 40C.
Add 20 ul 1N NaOH
Incubate 650C for 30 min
Add 20 ul 1N HCl
Purification of cDNA products:
1. Use QIAquick PCR Purification Kit, as per manufacturer
recommendations
2. Elute in 40 ul EB Buffer
3. Quantitate cDNA via spectrophotometry (OD260 = 1.0 equals 33 ug/ml
ssDNA)
10. Prepare Fragmentation mix (50 ul):
1. 5 ul 10X One Phor-All Buffer
2. X ul cDNA (5 ug)
3. Y ul DNase I (optimized for digesting cDNA to 50-200 nt @ 370C in 10
min ~ 0.6 U/ug of cDNA)
4. 45 ul – X ul – Y ul of Nuclease-free water
11. Incubate 370C for 10 min; 980C for 10 min
12. Prepare Labeling mix (60 ul):
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39 ul of Fragmentation product (~5 ug)
12 ul 5X Reaction Buffer (Enzo BioArray Terminal Labeling Kit)
6 ul 10X CoCl2 (Enzo Kit)
1 ul Biotin-ddUTP (Enzo Kit)
2 ul Terminal Deoxynucleotide Transferase (Enzo Kit)
13. Incubate 370C for 60 min; then add 2 ul 0.5 M EDTA
14. Store labeled-fragmented cDNA @ -200C until hybridization.
NOTE- We are currently working out the conc’s for spike-in’s & will provide you with
a cocktail/updated protocol when you are ready to label your RNA.