Functional Genomics Core
Diabetes and Endocrinology Research Center
Center for Molecular Studies in Digestive and Liver Disease
Microarray Sample
Preparation
Handbook
Director: Klaus Kaestner, PhD
Marie Scearce, PhD
John Brestelli
Saki Arsenlis
Location: 560 CRB
Phone: 215-746-6372
Fax:215-573-5892
Email: mscearce@mail.med.upenn.edu
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Table of Contents
BASIC EXPERIMENTAL REQUIREMENTS: ....................................................................................... 3
ISOLATION OF TOTAL RNA FROM TISSUE BY ACID/PHENOL EXTRACTION ....................... 4
REAGENTS: ................................................................................................................................................ 4
PROTOCOL:................................................................................................................................................ 4
DNASE TREATMENT: ............................................................................................................................... 5
ISOLATION OF TOTAL RNA FROM TISSUE USING TRIZOL AND QIAGEN RNEASY MINICOLUMNS .................................................................................................................................................... 5
RNA ANALYSIS .......................................................................................................................................... 6
MEASURING THE CONCENTRATION OF RNA: ............................................................................................ 6
DENATURING AGAROSE GEL: ................................................................................................................... 6
Reagents: ........................................................................................................................................... 6
Protocol: ............................................................................................................................................. 7
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Services:
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Comprehensive hybridization services…….$100/slide
Includes basic data normalization and analysis
PancChip version 2.2 …………………………$30/slide
Custom arrays …………………………………Arranged upon request
Basic Experimental Requirements:
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4 replicate samples for each experimental condition tested
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20ug of RNA (1-2ug/ul) per sample minimum (unless we are doing
amplification)
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RNA must be DNAse treated
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Copies of the denaturing agarose gel to check RNA integrity

OD 260 and OD 280 for each sample
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Isolation of total RNA from Tissue By Acid/Phenol Extraction
Reagents:
Denaturing solution
4M Guanidium thiocyanate, 0.1 M Tris /Cl pH 7.5 (can keep this
solution at room temperature)
Prior to use add -mercaptoethanol to a final concentration of 1%
Acid NaOAc solution
2M Sodium Acetate in DEPC-H20, pH = 4 in acetic acid
Protocol:
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For approximately 1 g of tissue (approximately a cubic cm), arrange 50 ml
Falcon tubes with 10 ml of denaturing solution in each on ice.
Dissect out tissue and place immediately into cold denaturing solution. And
immediately homogenize place homogenate on ice.
Finish homogenizing rest of samples before proceeding on.
Add 1 ml acidic NaOAc solution. Shake to mix.
Add 10 mL phenol (pH4) (1 ml per ml lysate). Shake and invert to mix
well.
Add 4 ml chloroform:IAA [24:1] (0.4 ml per ml of lysate). Shake
vigorously for 10 sec.
Incubate for 15-30 minutes on ice.
Centrifuge in falcon tubes at 6000 RPM for 20 minutes.
Remove the aqueous phase to a new microcentrifuge tube.
Add one volume isopropanol. Mix well by multiple inversion.
Incubate at –20C for 2 or more hours (overnight is fine).
Centrifuge at 6000 RPM for 30 minutes using Sorvall SLA-600TC.
Aspirate the supernatant carefully. The pellet should be visible at this
point.
Suspend the pellet in 0.5 ml denaturing solution, and transfer to 2
microcentrifuge tubes.
Add 50 L of acidic NaOAc to each. Mix well.
Add 550 ml of 1:1 phenol:chloroform. to each
Vortex and incubate 15 minutes on ice.
Centrifuge at 13,000 rpm and 4C for 10 minutes.
Remove the aqueous layer to a new microcentrifuge tube.
Add 550 L of chloroform:IAA to samples. Repeat steps 16- 18.
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Kaestner Lab
Add 550 L of isopropanol and mix well.
Incubate at –80C for 10 minutes (or at -20C for > 1 hour).
Centrifuge at 13,000 rpm and 4C for 10 minutes.
Aspirate the supernatant, and wash the pellet with 0.5 ml 70% ethanol.
Spin 3 minute
Aspirate carefully. Pulse in centrifuge to pull any remaining fluid to bottom
of the tube and aspirate and allow the pellet to dry for 10 minutes
under the hood.
Resuspend the pellet in 500 L of DEPC-treated H2O (1 ml for liver). If the
RNA is difficult to resuspend, incubate RNA at 60C for 1 hr then
vortex
DNase Treatment:
Reference: Wilson and Melton, Current Biology 1994, 4: 676-686.
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Resuspend RNA pellet (approximately 70-140 ug) in 70 ul of DEPC-treated
double distilled H2O
 10 l 10 x DNase buffer (400 mM Tris/Cl pH 7.9, 100 mM NaCl, 60 mM
MgCl2, 1 mM CaCl2)
 10 l 20 mM DTT
 2 l RNAsin, Promega 40U/l
 8 l (80 units) RNase free DNase (BM)
Incubate 30 minute at 37 oC
Extract with phenol/chloroform, and precipitate with 0.1 ml 7.5 M ammonium
acetate, 0.5 ml ethanol. Spin, wash with 70% ethanol, dry.
Isolation of total RNA from tissue using TRIZOL
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Homogenize tissue(at least 15sec) samples in 1 ml TRIzol Reagent
Add 20ug of Glycogen (may add up to 200ug for small concentration of
tissue) directly to the TRIzol
Add 200ul of chloroform per 1 ml of TRIzol Reagent. Shake tubes
vigorously by hand for 15 sec and incubate at RT for 3 min
Centrifuge for 30 min at 4 degrees Celsius 13K rpm.
Transfer to layer and add 0.5ml of isopropanol per 1ml of Trizol Reagent
used for the initial homogenization.
Incubate on ice for 10 min and spin for 25 min at 4oC
Wash the pellet with 1 ml of 75%ETOH, 25%DEPC water. Cf 25 min at 4oC.
Re-suspend pellet in 300 ul of TES (10mM Tris pH7.5, 1mM EDTA pH8,
0.1-0.2% SDS).
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Extract RNA with 600ul of phenol/Chloroform/isoamyl alcohol (25:24:1).
Vortex vigorously and spin 5 min at 4oC.
Carefully take the top phase, leaving any material at the interface. If there is
a large interface, repeat extraction step.
Add 1/10 volume, 3M Soduim Acetate pH7, and 3 volumes EtOH
Percipitate at -80oC until use (at least 2 hrs, preferably overnight).
Cf 30 min at 4oC
Wash the pellet with 1 ml of 75%ETOH, 25%DEPC water. Cf 25 min at 4oC.
resuspend to be >1ug/ul.
RNA Analysis
Measuring the concentration of RNA:
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Make a dilution of the RNA sample (usually 1:100) into TE buffer.
Use spectrophotometer to read OD 260
If the OD 260 if higher than 1.0 or lower than 0.1 make a new dilution to
bring the sample into a range where the accuracy of the readings is
greatest and retake the OD 260
Measure the OD 280 and the OD260/280 ratio. The OD 280 measures
contaminates in the sample. Therefore the OD260/280 ratio is an
indication of RNA quality. The OD 260.OD280 range for high quality
RNA is 1.8-2.0. Readings higher and lower indicate the presence of
contaminates in the RNA and significantly reduces the accuracy of
array analysis.
Scan the purified RNA prep in a spectrophotometer (in TE buffer) to detect
any phenol or protein contamination peaks. (Look for phenol peak at
270 nm)
Do not continue with cDNA synthesis if ratio is less than 1.6
Denaturing Agarose Gel:
Reagents:
1% Agarose Gel (1X TBE)
TE Buffer (10 mM Tris-HCl; 1 mM EDTA pH8)
Denaturing Sample Loading Buffer
2X TBE (pH8.3)
13%Ficoll (w/v)
0.01% Bromophenol Blue
7M Urea
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Protocol:
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Dilute 1-2 ug of RNA to a volume of 10 ul in DEPC water.
Add equal volume Denaturing Loading Buffer to RNA
Incubate RNA at 70 degrees Celsius for 10 minute; ice 5 minute
Add entire sample to Gel; Run 80 V for 1 Hour.
Integrity of RNA is assessed by visualizing 28S and 18 S Ribosomal
bands. Smeared bands indicate degradation; High molecular weight
bands indicate DNA contamination.
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