{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"submitter":["Pawel Sowinski"],"study_type":["transcription profiling by array"],"organism":["Zea mays"],"species":["Zea mays"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MEXP-1875"],"description":["This experiment evaluates quick (alarm) response to chilling in chilling-sensitive maize plants.
Maize inbred line cm109 were grown in optimal conditions until third leaf was fully developed.
At this stage plants were divided into three experimental variants: k0 - control plants, frozen
at the beginning of daylight, k4 - control plants kept in the same conditions and frozen after 4 hours
since beginning of daylight, c4 - plants kept in 14 deg. C for 4 hours since \"dawn\". At the mentioned
moments, leaves were harvested and frozen in liquid nitrogen for RNA isolation."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sample Processing - For chilling treatment, half of the plants were transferred to a cold chamber (14oC/12oC) at the beginning of photoperiod [a setup often used in molecular studies of maize response to low temperature ( Pimentel et al., 2005; Nguyen et al., in press)] for 4 h, and the other half kept as before served as controls.","Growth Protocol - Zea mays CM109 inbred line kernels were germinated in sand in darkness at 25oC and then plants were transferred to pots containing Knopï¾s nutrient solution supplemented with Hoaglandï¾s micro-nutrients. Further growth was conducted in a growth chamber under the photoperiod of 14 h/10 h and day/night temperature regime of 24ﾺC/22ﾺC, under the light irradiance of 250 ?mol quanta ï¾ m-2 ï¾ s-1, until the 3-rd leaf was fully developed.
(Parameters: time unit = seconds, temperature unit = C)","Labeling - 1
Solution Preparation
Preparation of cDNA Wash Buffer
2
Add 11.2 ml 100% ethanol (ACS grade or better) to the bottle labeled cDNA Wash Buffer. Mix
well and mark the label to indicate that the ethanol was added.
Preparation of aRNA Wash Buffer
Add 22.4 ml ACS grade 100% ethanol (ACS grade or better) to the bottle labeled aRNA Wash
Buffer. Mix well and mark the label to indicate that the ethanol was added.
Preparation of sodium carbonate buffer (200mM Na2CO3, 200mM NaHCO3): pH 9.0.
Dissolve 1.06g Na2CO3, and 0.84g NaHCO3 in 45 mL of RNAase-free water, and adjust pH to
9.0 with 12 N HCl; bring volume up to 50 mL with RNAase-free water. Aliquot into 0.5 mL
RNAase free tubes, and store at -20oC. Use one tube at a time, and discard the tube after use.
OR: Dissolve 1.06g Na2CO3 in 50 mL of RNAase-free water and 0.84g NaHCO3 in 50 mL of
RNAase-free water. Use one as the acid and one as the base. Combine the two until they reach
pH 9.0.
Preparation of Cy3 and Cy5 monoreactive dye. These dyes are supplied as five aliquots. The
dye in each tube is sufficient for four labeling reaction. Dissolve entire contents of a single tube
in 22 ï¾µL DMSO by flicking the tube several times, and leaving at RT for at least 30 min
protected from light. Spin at 1000 X g for 30 sec to collect the dye at the bottom of the tube. The
dye is now ready for use, but can be stored at -20oC for up to one month. Always protect the dye
from light by wrapping tubes with aluminum foil.
T7 Primer Annealing and First Strand cDNA Synthesis
T7 Primer Annealing
1. Place ~ 1.5 ï¾µg of total RNA into a sterile RNase-free 0.2 ml microfuge tube.
2. Add 1 ï¾µL of T7 Oligo (dT) Primer.
3. Add Nuclease-free Water to a final volume of 6 ï¾µL.
4. Incubate 10 min at 70ï¾°C in a thermal cycler.
5. Remove the RNA samples from the 70ï¾°C incubator and centrifuge briefly (~5 sec) to
collect sample at bottom of tube and immediately transfer to ice.
If your RNA samples are all at a standard concentration, you may want to assemble your T7
primer and H2O as a bulk mix for easy dispensing and increased accuracy.
First Strand Synthesis
Assemble the Reverse Transcription Master Mix at room temperature as a bulk mix and then
place on ice. It is good idea to make a master mix for several reactions as this reduces pipetting
errors. Mix well by gently pipetting up and down or flicking the tube a few times. It is prudent
to include 5% overage to cover pipetting errors. Use the table below for setting up multiple
reactions.
Number of Reactions First Strand cDNA
Master Mix 1 4 8 12 16
10x First Strand Buffer 1 ï¾µL 4 ï¾µL 8 ï¾µL 12 ï¾µL 16 ï¾µL
Ribo. Inhibitor 0.5 ï¾µL 2 ï¾µL 4 ï¾µL 6 ï¾µL 8 ï¾µL
dNTP 2 ï¾µL 8 ï¾µL 16 ï¾µL 24 ï¾µL 32 ï¾µL
Array Script 0.5 ï¾µL 2 ï¾µL 4 ï¾µL 6 ï¾µL 8 ï¾µL
Total Volume 4 ï¾µL 16 ï¾µL 32 ï¾µL 48 ï¾µL 64 ï¾µL
Total/Rxn 4.0 ï¾µL/Reaction
3
6. Transfer 4 ï¾µL of Master Mix to each sample, mix thoroughly by gently pipetting up and
down or flicking the tube a few times and place the tubes in a 42ï¾°C incubator. We
generally use PCR machine with lid temperature set for 48 C.
After the 2 h incubation at 42ï¾°C, centrifuge the tubes briefly (~5 sec) to collect the reaction at the
bottom of the tube. Place the tubes on ice and proceed to the second strand cDNA synthesis
(below).
Second Strand cDNA Synthesis
1. The reagent volumes are very small it is therefore recommended to make a master mix
for several reactions, and pipette 40 ul of second strand master mix directly into each first
strand reaction tube. Prepare the Second Strand Synthesis Master Mix on ice adding each
of the ingredients in the order listed in the table below. Gently mix by pipetting up and
down or by flicking the tube a few times, then centrifuge the tubes briefly (~5 sec) to
collect the reaction at the bottom of tube.
Number of Reactions Second Strand cDNA
Master Mix 1 4 8 12 16
DEPC H2O 31.5 ï¾µL 126 ï¾µL 252 ï¾µL 378 ï¾µL 504 ï¾µL
10x 2nd S. Buffer 5 ï¾µL 20 ï¾µL 40 ï¾µL 60 ï¾µL 80 ï¾µL
dNTP 2 ï¾µL 8 ï¾µL 16 ï¾µL 24 ï¾µL 32 ï¾µL
DNA Polymerase 1 ï¾µL 4 ï¾µL 8 ï¾µL 12 ï¾µL 16 ï¾µL
RNase H 0.5 ï¾µL 2 ï¾µL 4 ï¾µL 6 ï¾µL 8 ï¾µL
Total 40 ï¾µL 160 ï¾µL 320 ï¾µL 480 ï¾µL 640 ï¾µL
Total/Rxn 40 ï¾µL/Reaction
2. Incubate at 16ï¾°C for two hours in a thermal cycler or a refrigerated water bath and
proceed to cDNA Purification (below), or immediately freeze reactions at ï¾20ï¾°C. Do not
leave the reactions on ice for long periods of time.
cDNA Purification
Use the cDNA purification kit supplied with the Message Amp-II kit or the Ambion DNA Clear
Kit for cDNA purifications (Ambion Cat # 1756)
Before beginning the cDNA purification, preheat the 10 mL bottle of Nuclease-free Water to
50ï¾°C for at least 10 min.
1. Check that the cDNA filter cartridge is firmly seated in a 2 mL wash tube and pipet 50
ï¾µL cDNA binding buffer onto the filter in the cDNA filter cartridge.
2. Incubate at room temperature for 5 min. (DO NOT spin the cDNA binding buffer
through the cDNA filter cartridge).
3. Add 250 ï¾µL of cDNA binding buffer to each cDNA sample from the second strand
cDNA synthesis and mix thoroughly by repeated pipetting.
4. Pipet the cDNA sample/cDNA Binding Buffer onto the center of an equilibrated cDNA
Filter Cartridge.
5. Centrifuge for ~1 min at 10,000 x g, or until the mixture has passed through the filter.
4
6. Discard the flow-through and replace the cDNA filter cartridge in the 2 mL wash tube.
Make sure that the ethanol has been added to the bottle of cDNA Wash Buffer before
using it.
7. Apply 500 ï¾µL cDNA wash buffer to each cDNA filter cartridge. Centrifuge for ~1 min at
10,000 x g, or until all the cDNA wash buffer is through the filter.
8. Discard the flow-through and spin the cDNA filter cartridge for an additional minute to
remove trace amounts of ethanol.
9. Transfer cDNA Filter Cartridge to a cDNA Elution Tube. To the center of the filter in
the cDNA Filter Cartridge, apply 6 ï¾µL of nuclease free water that is preheated to 50ï¾°C.
Leave at room temperature for 2 min and then centrifuge for ~1.5 min at 10,000 x g, or
until all the nuclease-free water is through the filter.
10. Repeat the previous step with additional 6 ï¾µL of pre-heated nuclease-free water. The
double-stranded cDNA will now be in the eluate (~11 ï¾µL).
11. Discard the cDNA Filter Cartridge.
Check the cDNA concentration in the solution by applying 1.5 ul of eluted cDNA on to the
Nanodrop spectrophotometer. In general the cDNA yield should be around 5-10 ng / ï¾µL if you
start with ~1 ï¾µg of total RNA.
In Vitro Transcription to produce aminoallyl labeled cRNA
The oligo microarrays, being printed with positive-strand DNA elements, require labeled
negative-strand targets for hybridization. Since the first round of amplified aRNAs represents
the negativeï¾strand, it is recommended to label the aRNA itself. aRNA labeling can be done
using two methods: (a). direct incorporation of Cy-dye modified UTP during the process of in
vitro transcription, or (b). indirect labeling, by incorporating aminoallyl modified UTPs during in
vitro transcription followed by monoreactive cy-dye coupling. Since the cy-dye modified
nucleotides used for direct labeling are extremely expensive, we recommend the second
approach. Aminoallyl UTP (aaUTP) does not contain a bulky sidechain modification, which
means that one can replace 100% of the UTP with aaUTP during RNA synthesis without loss of
incorporation. We recommend using a 2:1 molar ratio of aaUTP to UTP. The ATP, CTP, GTP
Mix is assembled by mixing equal parts of the three nucleotides from 75 mM stock solutions so
that each nucleotide is at a 25 mM concentration. Below is table for preparing your aminoallyl
labeled cRNA master mix. Prepare the master mix on ice and mix well. Add 12 ul of master
mix to the ~8.0 ï¾µl of remaining double stranded cDNA
cRNA Synthesis Master
Mix Number of Reactions
1 4 8 12 16
aaUTP (50 mM) 1.5 ï¾µl 6 ï¾µl 12 ï¾µl 18 ï¾µl 24 ï¾µl
ATP, CTP, GTP Mix 6 ï¾µl 24 ï¾µl 48 ï¾µl 72 ï¾µl 96 ï¾µl
UTP Solution (75 mM) 0.5 ï¾µl 2 ï¾µl 4 ï¾µl 6 ï¾µl 8 ï¾µl
T7 10x Rxn Buffer 2 ï¾µl 8 ï¾µl 16 ï¾µl 24 ï¾µl 32 ï¾µl
T7 Enzyme 2 ï¾µl 8 ï¾µl 16 ï¾µl 24 ï¾µl 32 ï¾µl
Total Volume 12 ï¾µl 48 ï¾µl 96 ï¾µl 144 ï¾µl 192 ï¾µl
Total/Rxn 12 ï¾µl /Reaction
5
Mix the master mix and sample well with pipette, centrifuge at 3000 x g for 30 seconds. Incubate
the tube at 37oC in a PCR machine (the lid temperature should be set at 40oC). The minimum
recommended incubation time is 4 h, and the maximum is 14 h. We generally let our reactions
run overnight. Stop the reaction by adding 80 ï¾µL nuclease-free water to each cRNA sample to
bring the final volume to 100 ï¾µL. Mix thoroughly by gentle vortexing, and either proceed
directly to the cRNA purification step (below), or store at ï¾20ï¾°C.
aRNA Purification
Before proceeding to the dye coupling it is important to remove all the unincorporated
nucleotides from the aRNA. Check to make sure that each IVT reaction was brought to 100 ï¾µL
with nuclease-free water.
1. Add 350 ï¾µL of aRNA binding buffer to each aRNA sample, and proceed to the next step
immediately.
2. Add 250 ï¾µL of ACS grade 100% ethanol to each aRNA sample, and mix by pipetting the
mixture up and down three times. Do NOT vortex to mix and do NOT centrifuge.
3. Proceed immediately to the next step as soon as you have mixed the ethanol into each
sample. Any delay in proceeding could result in loss of aRNA because once the ethanol is
added, the aRNA will be in a semi-precipitated state.
4. Pipet each sample mixture from step 2 onto the center of the filter in the aRNA filter
cartridge. Centrifuge for ~1 min at 10,000 X g, or continue until the mixture has passed
through the filter.
5. Discard the flow-through and replace the aRNA filter cartridge back into the aRNA
collection tube.
6. Apply 650 ï¾µL wash buffer to each aRNA filter cartridge, centrifuge for ~1 min at 10,000
X g, or until all the wash buffer is through the filter.
7. Discard the flow-through and spin the aRNA filter cartridge for an additional ~3 min to
remove trace amounts of wash buffer.
8. Transfer filter cartridge(s) to a fresh aRNA collection tube, to the center of the filter, add
30ï¾µL nuclease-free water (pre-heated to 50C).
9. Leave at room temp for 2 min and then centrifuge for ~1.5 min at 10,000 X g, or until the
nuclease-free water is through the filter. Repeat with 30ï¾µL of nuclease-free water.
10. The aRNA will now be in the aRNA collection tube in ~60 ï¾µL of nuclease-free water.
Determine the concentration of RNA using the Nanodrop or a conventional spectrophotometer.
Total yield of aminoallyl labeled cRNA is generally in the range of 20 to 40 ug of cRNA.
Typically we use about 3 ug of cRNA target per slide or 6 ug for the two slide set. Aliquot ~6 ï¾µg
of aRNA for dye coupling and completely dry it using a Speedvac centrifuge set at room
temperature. Store the remaining aRNA at -80 for further use.
Coupling AA-cRNA to Cy Dye Ester.
1. Dissolve the dried cRNA with 5 ï¾µL of NaHCO3 buffer by flicking the tube several times
and leaving the tube at RT for at least 20 min.
2. Add 5 ï¾µL of Cy3 or Cy5 (in DMSO) to each tube, and mix them thoroughly by flicking
the tube several times.
3. Spin the tube at 1000 X g for 30 sec.
4. Incubate the dye and cRNA mix in the tube at RT for 2 h covered in aluminum foil.
Quenching Reaction This optional step involves quenching any unreacted Cy dye by adding an
excess of primary amines.
1. Add 4.5 ï¾µL 4M hydroxylamine.
2. Incubate for 15 min in the dark at RT.
Removal of Unincorporated Dye The Qiagen RNeasy MinElute column is used for this
purpose.
1. Adjust sample to a volume of 100 ï¾µL with RNAase-free water. Add 350 ï¾µL of RLT
buffer, and mix thoroughly.
2. Add 250 ï¾µL of 96ï¾100% ethanol to the diluted RNA, and mix thoroughly by pipetting.
Do not centrifuge, continue immediately with step 3.
3. Apply 700 ï¾µL of the sample to an RNeasy MinElute Spin Column in a 2 mL collection
tube (supplied). Close the tube gently, centrifuge for 15 s at 8000 x g, and discard the
flow-through.
4. Transfer the spin column into a new 2 ml collection tube. Pipet 500 ï¾µL RPE buffer onto
the spin column. Close the tube gently, and centrifuge for 15 s at 8000 x g to wash the
column. Discard the flow-through (reuse the collection tube in step 5). Note: RPE buffer
is supplied as a concentrate; ensure that ethanol is added before use.
5. Add 500 ï¾µL of 80% ethanol to the RNeasy MinElute Spin Column. Close the tube
gently, and centrifuge for 2 min at 8000 x g to dry the silica-gel membrane. Discard the
flow-through and collection tube. Repeat with an additional 500 ï¾µL of 80% ethanol.
6. Transfer the RNeasy MinElute Spin Column into a new 2 mL collection tube (supplied).
Open the cap of the spin column, and centrifuge in a microcentrifuge at 12000 x g for 5
min. Discard the flow-through and collection tube.
7. To elute, transfer the spin column to a new microfuge tube. Pipet 20 ï¾µL DEPC water and
leave at RT for 2 min. Close the tube gently, and centrifuge for 1 min at 12000 x g for 1
min.
8. Repeat step 7 with an additional 20 ï¾µL of DEPC water. (The labeled RNA may also be
eluted using only 10 ï¾µL of DEPC water if a higher concentration is desired).
Measure the amount of dye incorporated into aRNA using a NanoDrop or conventional
spectrophotometer. We generally recover enough dye labeled cRNA (~5 ug) for two slide
hybridizations.
(Parameters: Amount of nucleic acid labeled = 5, Amplification = PCR, Mass unit = Micro gram)","Hybridization - www.maizearray.org protocol http://www.maizearray.org/files/Hybridization_Protocol_For_cRNA_Targets.pdf Hybridization with cRNA Targets Overview: This hybridization protocol has been optimized for use with cRNA targets and 70-mer oligonucleotide probes. It is an adaptation of a protocol developed for cDNA arrays produced by the Potato Functional Genomics Project (http://www.tigr.org/tdb/potato/microarray_SOPs.shtml). With practice, this protocol yields very reproducible images with low back ground and high signal to noise ratio. Materials - 20X Saline-Sodium Citrate (SSC) - 10% Sodium Dodecyl Sulfate (SDS) - Bovine Serum Albumin (BSA) - Formamide, redistilled - Isopropanol - Ethanol - Coplin jar - Microarray holder and wash station - tRNA (10 mg/ml) - Salmon Sperm DNA (10mg/ml) - Microscope Lifter Slip - Hybridization chamber - 1 L .22 ï¾µm CA (cellulose acetate) Filter System Methods DNA Probe Immobilization: Re-hydration and UV cross linking can be done well in advance before microarray hybridization, and the slides can be stored at room temperature for several months. However we do not recommend storage of washed microarray slides for extended periods of time. 1. Mark the corner boundaries of the array on a separate glass slide. Once spots have been immobilized and the slide is washed, the spots will not be visible (the spots are only visible due to the presence of SSC crystals). One needs to know the boundaries of the array in order to correctly place coverslip over array during hybridization. 2. Re-hydrate slide over a 55ï¾°C water bath for ~5 seconds. Hold slide label side down over the water vapor. Watch spots carefully so that they do not over-hydrate and begin to merge together. In humid environments this is particularly important. 3. Snap dry the slide on a 45ï¾°C heating block for 5 seconds. Place slide label side up on heating block. Allow slide to cool for 1 minute or cool with compressed air by blowing over the back of the slide. 4. Repeat steps 1-3 a total of four times. 5. UV cross-link the slides by exposing them in batches, label side up, to 180 mJ in a commercial cross-linker (we employ a Stratalinker). 6. Wash the slide in 1% SDS for 5 minutes at room temperature in wash station with stir bar rotating at ~120 RPM. Remove SDS by dipping the slides ten times into DDH2O. 7. Immediately transfer the slides to 100% ethanol, dip five times, then incubate for three minutes with shaking. 8. Spin dry slide in centrifuge at no more than 200 x g for 2-4 minutes. Pack bottom of 50 mL centrifuge tube with Kimwipes. Using forceps carefully place slide into tube with label at the bottom. Repeat spin if any liquid is remaining on the slide surface. 9. Repeat the ethanol wash if any visible streaks remain after spin dry. Store slide in a lint-free light-proof box at room temperature with low humidity. Prehybridizaton 1. Prepare prehybridization buffer (5X SSC, 0.1% SDS, 1% BSA) and sterilize by filtration using a CA filter. Preheat ~50 ml to 42ï¾°C in a coplin jar for 30 minutes. The prehybridization buffer can be made up in advance and stored at room temperture 2. Place the printed slide(s) to be used for the hybridization in a Coplin jar containing prehybridization buffer preheated to 42ï¾°C. Incubate at 42ï¾°C in water bath for 45 minutes. 3. Wash the slides for 5 minutes in the wash station filled with room temperature ddH2O. 4. Repeat step 3 with fresh ddH2O for 5 minutes. 5. Shake the slides at room temperature in ethanol for 2 minutes. 6. Dry down in centrifuge by placing slides in slide rack on a swinging plate tray (500 rpm for 5 minutes). If you see white streaks on the slide repeat water/water/ethanol wash cycle. 7. Use slides immediately following pre-hybridization to ensure optimal hybridization efficiency. Hybridization 1. Prepare 1X hybridization buffer (50% formamide, 5X SSC, and 0.1% SDS). To prepare 1.0 ml of 1x hybridization buffer: 250 ï¾µl 20X SSC, 10 ï¾µl 10% SDS, 500 ï¾µl formamide, 180 ï¾µl H20, 40 ï¾µl tRNA (10 ug/ï¾µl), and 20 ï¾µl Salmon Sperm DNA (10 ug/ï¾µl). 2. Resuspend labeled target (Cy3/Cy5 probe mixture) in ~60 ï¾µL of 1X hybridization buffer or 130 ï¾µl for the MOA and MOB slide set. The dried target can be prepared either by speed vacuum or ethanol precipitation. Use about 3 ug of each cRNA target per slide or 6 ug of each target for the two slide set. The target may be hard to get into solution but will go into solution after the heating step below. 3. To denature, heat the probe mixture at 95ï¾°C for 3 minutes and snap cool on ice for 30 sec. 4. Centrifuge the probe mixture at maximum angular velocity for 2 minute to remove any particulates. Keep at room temperature or warm slightly and use immediately. Note: Expose Cy labeled probe to light as little as possible during the hybridization and washing process. Applying the Labeled Probe Mixture 1. Rinse ArrayItï¾ Hybridization Cassette with distilled water and dry thoroughly. 2. Make sure flexible rubber gasket is seated evenly in gasket channel. 3. Insert the microarray (1\" x 3\" or 25mm x 75mm slide) into cassette chamber with the DNA (labeled side) side up. 4. Place the lifter slip over the microarray slide (make sure the white stripe of the lifter slip is at the lower side) 5. Apply the PRE-HEATED sample slowly to the one end of the lifter slip and let it disperse. Use 50 to 60ï¾µl of sample for each slide being careful not to introduce air bubbles. 6. Add 25 ï¾µl water to the lower groove inside the cassette chamber. 7. Quickly place the clear plastic cassette lid on top of the cassette chamber. 8. Apply downward pressure and manually tighten (clockwise) the four sealing screws. 9. Check all four screws again to confirm a tight seal. 10. Place the cassette into a hybridization oven set at 42ï¾°C. 11. Allow the hybridization reaction to proceed for 12 to 14 hours. 12. After hybridization, remove cassette, manually loosen the four sealing screws (counterclockwise) and remove lid. 13. Remove the microarray slide from the cassette chamber using forceps and place the slides into the washing station containing first wash (see below). Note: Do not flip the hybridization chamber upside down during hybridization; this may cause the lifter slip to shift from the slide and adversely affect the hybridization. Microarray Washing 1. Wash slide in the following solutions for 5 min each: 1. 2x SSC, 0.1% SDS @ 42ï¾°C 2. 0.1x SSC @ room temperature 3. 0.05x SSC @ room temperature 4. 0.05x SSC @ room temperature 2. Washing is done by immersing the slides in a glass the Telechem wash station (Cat# HTW) containing approximately 450 ml of wash buffer, followed by placing it on a magnetic stir plate set at ~120 rpm. Pre-heat the first wash solution to 42ï¾°C, and make sure the slides are completely immersed in wash buffer. To ensure even washing, rotate the slide holder 90 degrees mid way through each wash. 3. After completion of the washes, spin dry the slide in the centrifuge at no more than 1000 rpm for 2-4 min. a. Pack bottom of 50 mL plastic disposable centrifuge tube with Kimwipes. b. Using forceps, carefully place slide into tube with label at the bottom. c. Repeat spin if any liquid remains on the slide. Note: Washing is a critical step and care needs to be taken not to over or under wash your slides. For consistent results it is essential to wash the slides the same way each time.
(Parameters: Chamber type = OTHER: ArrayItï¾ Hybridization Cassette , Quantity of label target used = 6, Mass unit = Micro gram, time = 12, Tiny time unit = hours, Volume = 50, Volume unit = Micro litre, temperature = 42)","Nucleic Acid Extraction - www.maizearray.org trizol extraction protocol http://www.maizearray.org/files/RNA_Isolation_Using_Trizol_And_Qiagen_RNAeasy_Columns.pdf
Total RNA Isolation Using Trizol and Qiagen RNAeasy Columns
Overview
This protocol is quick and works very well for preparing 20 to 40 ug of very clean, salt free, RNA. The RNA prepared from this protocol is ready for target preparation using the Ambion Message Amp II procedure to produce aminoallyl labeled cRNA.
Materials Required
RNAase-free mortar and pestle: cover the mortar and pestle with aluminum foil and bake at least 3 hours at 180ï¾°C
RNAase-free 1.5 or 2.0 mL (preferred) microfuge tubes
Liquid nitrogen
Microfuge
RNAase-free pipette tips
Qiagen RNAeasy Mini elute columns and buffers (Qiagen Cat # 74204)
DEPC-treated H2O
Trizol (Invitrogen)
Procedure
1. Homogenize tissue in liquid nitrogen. It is not necessary to homogenize large amounts of tissue as the Qiagen RNAeasy columns can only bind ~40 ug of RNA and overloading the column is not advised. If you are working with pooled samples you may find that you
have more ground sample than you can use. It is convenient to have a small measuring device (teaspoon, grooved spatula, etc) to transfer the ground material directly to the Trizol in the microfuge tube.
a. Chill mortar with ~100 mL of liquid nitrogen.
b. Add frozen tissue after nitrogen is nearly completely evaporated.
c. Grind tissue quickly but carefully.
d. When liquid has fully evaporated, grind faster to produce a fine talc-like powder.
2. Add 1/8 to 1/4 teaspoon to 1.0 ml of Trizol. It is important to mix well immediately by vortexing and not allow tissue to thaw that is not in contact with the Trizol. You may want to prewarm the Trizol (35-40 C) so that it does not freeze when it comes into
contact with the frozen tissue. This is the most critical step in the procedure as once the tissue is completely mixed with the Trizol, it is protected from RNAases.
3. Incubate for five minutes at room temperature (RT), votexing frequently.
4. Add 0.2 ml of chloroform to the Trizol, and vortex for 15 seconds.
5. Incubate for 1 minute at RT, vortex again for 15 seconds.
6. Centrifuge at 15,000 xg for 10 minutes to separate phases
7. Remove 200 ul from the top layer and add to 700 ul of Qiagen RLT buffer in a new tube. Remove the rest of the top layer and freeze at -20 to serve as a backup in case your initial yield is low.
8. To the 200 ul of sample now combined with 700 ul RLT buffer, add 500 ul of 96-100% ethanol. Mix well by vortexing but do not centrifuge.
9. Apply half of your sample (~700 ul) to a Qiagen MinElute spin column placed in a 2 ml microfuge tube. Spin 15 seconds at ~10,000 rpm. Discard flow through and repeat procedure with the second half of your sample.
10. Remove the Minelute column to a new 2 ml microfuge tube and add 500 ul of RPE to the spin column. Spin 15 seconds at ~10,000 RPM.
Discard flow through.
11. Add 750 ul of 80% ethanol and spin at ~10,000 rpm for 15 seconds. Repeat this step a second time with another 750 ul of 80% ethanol. This step is repeated to ensure removal of all guanidine salts that may inhibit downstream applications.
12. Transfer the Minelute spin column to a new 2 ml microfuge tube. Spin for 5 minutes at top speed with the cap off. This ensures the removal of trace amounts of ethanol that may interfere with downstream applications.
13. To elute RNA, transfer spin column to a new, 1.5 ml microfuge tube. Elute with 10 ul of RNAase free water. Repeat with another 10 ul of RNAase free water. If you suspect low RNA concentration, you may elute with 12 ul of RNAase free water. It is desirable to
have a concentration of ~ 1 ug/ul if possible. Check concentration on a gel or spectrophotometer.
(Parameters: Extracted product = total_RNA, Amplification = PCR)"],"figure_sub":["MIAME Score","Raw Data","Organization","Assays and Data","Additional Files","MAGE-TAB Files","Array Designs"],"pubmed_authors":["Pawel Sowinski"],"additional_accession":[]},"is_claimable":false,"name":"Transcription profiling of maize to identify gene expression changes after chilling for 4 hours (alarm phase)","description":"This experiment evaluates quick (alarm) response to chilling in chilling-sensitive maize plants.
Maize inbred line cm109 were grown in optimal conditions until third leaf was fully developed.
At this stage plants were divided into three experimental variants: k0 - control plants, frozen
at the beginning of daylight, k4 - control plants kept in the same conditions and frozen after 4 hours
since beginning of daylight, c4 - plants kept in 14 deg. C for 4 hours since \"dawn\". At the mentioned
moments, leaves were harvested and frozen in liquid nitrogen for RNA isolation.","dates":{"release":"2009-11-01T00:00:00Z","modification":"2022-11-21T23:03:02.791Z","creation":"2022-03-09T13:12:09.688Z"},"accession":"E-MEXP-1875","cross_references":{"EFO":["EFO_0002768"]}}