Protocol 1.1 Total RNA extraction from mammalian tissue/cells
Originally created by the Sanger Centre, modified by J.Hu
Reagents
•TRIZOL reagent (Gibco BRL,Cat#15596-026)
•Homogeniser(Janke&Kunkek,IKA-labortechnik,Ultra-turrax T25)
•DNase, RNase free water
•Chloroform, Isopropanol, Ethanol: All from Sigma
Procedure
1.Remove tissue vial from liquid nitrogen and place contents in petri dish.
Allow frozen sample to warm up slightly (20-30 seconds at RT) to make
cutting easier. Hold sample with forceps and chop up as small as possible
using scalpel.chill the pestle and mortar with liquid nitrogen.
2.Transfer the chopped tissue into the chilled mortar (scrape off sides with
scalpel). Crush to a powder-like consistency with pestle, using paper towel
to prevents loss of sample from mortar.
3.Dispense the powder into the pre-weighed 50ml Falcon tube and
immediately re-weigh the tube. Calculate the weigh of pulverised sample
and add 1ml of TRIZOL reagent per 50mg of tissue/or 107 cells. Place tube
on ice.
4.Wash the homogeniser twice with dist.water,3 times ethanol and once with
TRIZOL reagent at the start and between the samples. Homogenise with
3x10 second bursts with 60 seconds cooling between bursts. Keep samples
on ice bucket during this process. When finish using, wash homogeniser
twice with water and once with methanol. In addition, may require boiling
water,SDS.(The tissue homogeniser should be clean and RNase-free)
5.Incubate homogenised samples at room temp. for 5 min.
6.Split homogenate into 1ml aliquots in 1.7ml eppendarf tubes and add
0.2ml of chloroform in it. Mix by vortex for 15 sec. and incubate at room
temp. for 2-3 min.
7.Centrifuge samples at 10,000rpm for 15 min at 4°C.
8.Transfer aqueous upper phase to another 1.7ml eppendarf microcentrifuge
tube and add 0.5ml of isopropanol per 1ml aqueous and mix by inversion.
9.Incubate at room temp. for 10 min and centrifuge at 10,000rpm for 15 min
at 4°C. The RNA pellet should now be seen at the bottom of the tube.
10.Remove supernatant carefully and wash pellet once with 1ml of 75%
ethanol, vortex and centrifuge at 6,500rpm for 5min.
11.Remove supernatant and air-dry RNA pellet for 5min, do not over dry the
pellet. Resuspend in about 100-200ul DNase, RNase free water to dissolve
the pellet or incubate at 37°C until pellet is completely dissolved.
12.Quantitate total RNA using UV spectrophotometer (reading at
A260),polyA selection can then be performed on total RNA using oligotex
mRNA kit from QIAGEN.
Quality and quantity control of RNA samples:
Gel: 1% Agarose in 0.5x TBE(RNAse-free,PH 7.8)
Running buffer: 0.5x TBE(RNAse-free,PH 7.8)
2x gel loading buffer: 95% formamide
18mMEDTA
0.025%SDS
0,025%Bromophenol blue
• 2µg RNA+loading buffer
•Boil at 95°C for 3 min
Protocol 1.2 mRNA purification batch protocol By QIAGEN Oligotex
Midi kit(Cat#700420)
Before starting:
•Set up heat blocks at 37°C and 70°C
•Heat Oligotex suspension to 37°C, mix by vortexing and then place at room
temperature
•Heat buffer OEB at 70°C
•Determine the quantity of total RNA by measuring the absorbency at
260nm(A260)
•If buffer OBB form a precipitate, warming at 37°C and then place at room
temperature
•Unless otherwise indicated, all steps performed at room temperature
Steps:
1.Upon the amount of total RNA has been measured (do not use ≥3mg total
RNA), adjust the volume by nuclease-free water accordingly and add buffer
OBB and Oligotex suspension appropriately (mix thoroughly by pipetting):
Total RNA water to Buffer OBB Oligotex suspension
≤250µg 250µl 250µl 15µl
250-500µg 500µl 500µl 30µl
500-750µg 500µl 500µl 45µl
2.Incubate the mixture at 70°C for 3 mins
3.Remove the tubes from heat and incubate at room temperature (20-30°C)
for 10 min.
4.Centrifuge at 13,000g for 2 min and carefully remove the supernatant by
pipetting (do not over pipetting the supernatant in case of losing Oligotex
resin)
5.Resupend the Oligotex: mRNA pellet in 1ml buffer OW2, mix by
vortexing and then spin at 13,000g for 2min,and carefully remove the
supernatant by pipetting.
6.Repeat the above step once.
7.Add 20-100µl hot (70°C) buffer OEB (with multiple sample, place the
tube on 70°C block to ensure buffer OEB is hot), resuspend the resin by
pipet up and down 3-4 times and spin at 13,000g for 2min, and then
carefully transfer the supernatant to a Rnase-free tube (mRNA is ready)
8.Reapeat the above step once to combine the elution.
The standard QIAGEN protocol can be found on the QIAGEN website:
www.qiagen.com
Protocol 2:Generation of a Bacterial mRNA “cocktail”
Reagents
•cDNA clone for the B.subtilis trp gene (ATCC,87485)
•cDNA clone for the B.subtilis lysA gene (ATCC,87482)
•cDNA clone for the B.subtilis thrB gene (ATCC,87484)
•cDNA clone for the B.subtilis dapB gene (ATCC,87486)
•cDNA clone fir the B.subtilis pheB gene (ATCC,87483)
•MegaScript T3 in vitro transcription (IVT) kit (Ambion, Cat#1338)
•DEPC dd water
•RNase I (10ug/ul)(Sigma, Cat#R-6513)
•Phenol/chloroform (1:1)
•Restriction endonuclease NotI (10U/ul)(New England Biolabs, Cat#189S)
•1% agarose 1xTBE gel (made up with DEPC H2O)
•100% and 75% ethanol (the later made up with DEPC H2O)
Steps
1. Isolate plasmid DNA from each of five B. subtilis cDNA clones by
QIAGEN plasmid preparation kit. The suggested template concentration is
0.5ug/ul in dH2O or TE.
2. Digest 5ug of each plasmid DNA by NotI to get linearized DNA.
3. Generate in vitro mRNA transcripts of each gene using the MEGAscript
in vitro transcription (IVT) kit (detail process were described in
manufacture’s protocol). Dissolve purified IVT mRNAs in DEPC ddH2O.
4.Quantitate IVT mRNAs using UV spectrophotometer reading at A260 and
assess quality by electrophoresis of 2ug RNAs on a 1% agarose TBE gel.
5. Add 3x volumes of 100% ethanol to aqueous RNA samples.
6.To make 250ul 10x stock of the bacterial mRNA”cocktail” in 75% ethanol
by combining each of five mRNAs to acquire the
final concentrations in the mixture are as follows:
lysA=0.3ng/ul
pheB=1.5ng/ul
thrB=3ng/ul
dapB=15ng/ul
trp=30ng/ul
7.Dilute down to 1x working solution using 75% ethanol.
Protocol 3. Generation of fluorescently-labelled single-strand cDNA
Target by CyScribe first-Strand cDNA labelling kit
Reagents
•CyScribe first-strand cDNA labelling kit (Amersham, Cat#RPN6200)
•dCTP-Cy3,dCTP-Cy5(1mM stock,Amersham,PA53021,PA55021)
•1M NaOH
•1MHCL
•3M NaAc, PH 5.2
•AutoSeq• G-50 spin columns (Amersham Pharmacia Biotech,Cat#275340-02)
Before start
•Set up heat block at 70°C,42°C and 37°C
•Place the required reagents excluding enzyme on ice to thaw, mix and
briefly spin down
Steps
1.Co-precipitate1µl of bacteria mRNA and 1ug of sample mRNA by adding
1/40 vol. 3M NaAC, incubate at -70°c for 30min.
2.Centrifuge at 13,000g for 10min,wash the pellet with 100µl 75%Ethanol
and dry at RT for 10min.
3.Resuspend mRNA in: 9µl Nuclease free water
1µl Anchored oligo (DT)
1µl Random nonamers
11µl total
Mix the above well by pipetting up and down, then incubate the mixture at
70°C for 5min,and then let the reaction cool at RT for 10min to allow the
primers to anneal with mRNA template, and then briefly spin for 30sec to
bring the reaction down to the bottom of the tube.
4. Place the tube on ice and add the following reagents to it in order:
5x CyScribe buffer 6µl
0.1M DTT 3µl
dNTPs (for dCTP dye) 1µl
Nuclease free water 4µl
dCTP-Cy3 or Cy5 3µl
CyScribe RT 2µl
Total 30µl
Mix the reactions by vortexing and spin them for 30sec
5.Incubate the reaction at 42°c for 1.5hours,When finished, put the tube on
ice.
6.Add 1.5µl of 1M NaOH and incubate at 70°C for 20min to hydrolyse the
RNA
7.Add 1.5µl of 1M HCL to neutralise the reaction.
8.Remove the short oligomers(mRNA) and unincorporated nucleotides by
AutoSeq•G-50 columns(follow the manufacture’s instruction).Once
labelled ss cDNA has been purified through the g-50 columns, the resulting
flow-through should be approximately 33µl and be faintly coloured pink or
blue depending on which Cy dye has been incorporated.
Protocol 4. Competitive Hybridisation of labelled ss cDNAs onto
Microarrays
Originally created by the Sanger centre, modified by J.Hu
Reagents
•Labelled ss cDNA pellet derived from RNA sample 1& 2
•Hybridisation buffer (5x SSC, 6x denhard’s solution, 60mM Tris HCL, PH
7.6,0.12% sarkosyl, 48% formamide; filter sterilised)
•Human Cot-1 DNA (2ug/ul, Gibco BRL,Cat#15279-011)
•PolyA DNA (2ug/ul, Sigma,Cat#P0887)
•Wash solution 1 (2x SSC)
•Wash solution 2 (0.1 x SSC, 0.1% SDS)
•Wash solution 3 (0.1 x SSC)
•Ethanol(70% and 100%)
•48 quadrant microarray slide
•Cover slip(25X60mm,BDH,Cat#406/0188/87)
•Humid hybridisation chamber(moistening with 5mls solution:40%
formamide,2XSSC)
Steps
1.Combine the two ss cDNA samples as follows:
33ul ss cDNA sample 1(Cy3-labelled)
33ul ss cDNA sample 2(Cy5-labelled)
4ul human Cot-1 DNA
4ul polyA
7µl 3M NaAc,PH5.2
218µl 100%ethanol
293µl total
2.Precipitate ss cDNa,polyA and Cot-1 DNA at -70°c for 20min.
3.Centrifuge at full speed, 10min to pellet sample and wash briefly in 70%
ethanol, then air dry pellet thoroughly.
4.Resuspend the pellet in 40µl hybridisation buffer and add 8µl ddwater to
bring the total volume to 48µl.
5. Place the above mixture in 100°c for 5 mins, and let it cool down to room
temperature in about 10 mins. Spin briefly to bring the evaporated liquid
down to the bottom of the tube.
6.Pre-cool the coverslip and microarray slide to 4°c for 30min.Carefully
apply the sample in hybridisation buffer onto the centre of a coverslip placed
on a flat surface.
7.Hold the slide containing the microarray face down, Gently lower the slide
onto the coverslip so that the area containing the array is positioned entirely
within the coverslip. (Note: once the hybridisation mixture makes contact
with the lowering slide, it will spread evenly across the microarray, small air
bubbles will gradually disappear during the hybridisation.)
8.Place microarray slide DNA side up in humid chamber with humidity
maintained by 2X7 cm 3MM paper moistened with 5mls of
40%formamide,2XSSC. Incubate at 47°c for 12-24 hours. (make sure the
chamber was sealed with autoclave tape).
6.Remove microarray out from humid chamber and quickly place in slid
rack submerged in 100-200ml of RT wash solution 1. (Note: the coverslip
should slide off itself easily), wash at RT in appropriate volume for 5 mins
with gentle shaking.
7.Transfer to wash solution 2 for washing 30 mins at RT. Repeat washing at
wash solution 2 for another 30 min.
8. Transfer to wash solution 3 for washing 5 mins at RT.
(All the washing steps should be protected from the light.)
9.Quickly transfer the slide in the rack to be able to centrifuge at 1000rpm
for 2 min to dry the slides (Important note: the Sorvall RT 60000D
centrifuge or equivalent should carry out this step).
10.slides now are ready for scanning. (Sanger Centre recommends using GSI
Lumonics laser-based scanner, scanning at the two wavelengths compatible
with efficient excitation for Cy3 and Cy5 and acquiring both images).
Protocols for scanning microarray slides using the GSI-Lumonics
SA4000 and quantifying microarray images using GSI-Lumonics
QuantArray
Part I: Scanning microarray slides by GSI-Lumonics SA4000
A GSI-Lumonics SA4000 scanner is controlled by the GSI-Lumonics
ScanArray software, which is used for image acquisition.
Scanner set-up
After switching on the scanner and starting the ScanArray software, the
correct instrument (SA4000 has to be selected.laser1 and laser 3 are required
for Cy3 and Cy5 flurophors and can be switched on using the buttons in the
menu bar. Warm-up time for the laser to full and stable power is 15 min and
is indicated on the screen.
Loading the slide
Insert a slide with the DNA side face up, and then retract the sample holder
using the Eject icon on the toolbar
Image acquisition
Once the slide is load, Click Start on the Acquire menu. Select the desire
protocol, which means that the laser has to be focussed on the plain of the
fluorophors for the maximal signal. Specify the acquisition area by entering
the X and Y positions and the width and height in the appropriate fields. A
Quick Scan at 50microns should be performed to set the laser strength and
PMT gain. A typical setting is 75-85 for Cy3 and 65-80 for cy5 for both
laser intensity and PMT gain. The final image is acquired with resolution of
10microns and the image is automatically saved in TIFF format in the
selected folder.
Image storage
Images are either transferred to CD-ROM or stored in the server.
Part II: Quantifying microarray images using GSI-Lumonics QuantArray
Requirements
TIFF images acquired with ScanArray. For data normalisation and
background correction, the QADataReduce macro is needed to be placed in
the templates/spreadsheet solutions folder of MS office.
Performing an Analysis
Setting up the experiment: Select the desired protocol and click open. The
protocol determines the correct array layout and the path to the right
Clonetracker annotation file for each array batch. Then open images by
opening Cy3 first and then Cy5. Contrast can be adjusted by clicking on the
contrast icon in the menu and should be set to around 80.Select Register
images to align the two images together. And then click Specify location,
the upper left corner of the array (Cy3/Cy5 spot) should be chosen as marker
and in Edit Pattern,
The grid can be adjusted manually to the array. In Locate Spots,
choose the Use Nominal locations, click the Start Locate,
the software automatically determines the exact location of the spots. To
ignore a given spot, right click on the spot and select ignore, An X will be
placed through the selected spots.
Quantification the spots: Once the locate Spots step is complete,
QuantArray is ready to quantitate the spots signal. When the View Report
step is active, you can quickly determine the intensity and the morphology of
a single spot on each image by double clicking on the desired spot in an
image. For the array pattern used in thee Hinxton arrays, the “Histogram
method” was proven useful. In this method, the highest 60-95% of the pixels
are used as signal and the lowest 5-30% as background. The analysis results
can be visualised by double clicking on a spot or by choosing the report
buttons (scatter plot, bar graph etc.).
Exporting the data: Use the Export data step, the Open Excel with last
exported file button will be activated. This button will automatically
import the file that has just been exported into Excel .
Normalisation and background correction: The QAData Reduce macro
can perform Background Subtraction which reports the intensity of each
spot in the array minus the background for that spot and Normalisation
which corrects the intensity of each spot for variations in the overall
intensity of the 2 images (Cy3 and Cy5). The background subtraction and
normalisation can be performed on a set of images in one operation. From
the Microsoft Excel mainwindow, select File/New/spreadsheet
solution/Normalise,click Enable Macros. For most experiments,
normalisation to total intensity of the whole array should be chosen. After
applying the macro, signal intensities of the two channels are automatically
normalised and background subtracted and placed into a new spreadsheet
that can be used for further data analysis.