Labeled cDNA Probe Generation from Yeast RNA

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Cell & Molecular Biology Laboratory
BIOL 406
cDNA Probe generation for Yeast Microarrays
1
Labeled cDNA Probe Generation from Yeast RNA
Objectives:
1. Use Total Yeast RNA to artificially synthesize labeled cDNAs for hybridization in microarrays.
2. Understand the function of Reverse Transcriptase, Primers & use of Fluorescent tags
3. Handle light sensitive reagents properly
4. Understand how the method of labeling cDNAs can affect final signal observed in microarrays
5. Work with limited reagents and small volumes using extreme caution
6. Understand the steps required in experimental design and planning
READ THIS PROTOCOL CAREFULLY. MAKE SURE YOU ARE
PREPARED TO ASK ANY TECHNICAL OR THEORETICAL QUESTIONS
BEFORE WE BEGIN THIS EXPERIMENT. IF IN DOUBT ABOUT
ANYTHING, ASK!
Probe Generation from Total Yeast RNA
We will use the 3DNA Kit from Genisphere to synthesize our labeled cDNAs. To measure
changes in the mRNA levels in Yeast during the diauxic shift, we need to have a baseline level of
gene activity (i.e. mRNA levels). The initial time point (9 hours) with excess glucose in the media
will be our reference level of mRNAs. We will prepare labeled cDNA by reverse transcription
using special primers that will bind the fluorescent dyes Cy3(green) or Cy5(red). The cDNAs will
be hybridized to the microarrays in next week’s lab.
To ensure that we measure accurate changes in mRNA levels, we will label cDNA from the initial
time point (reference) with Cy3 (green), and cells at each successive time point (13, 17 and 21
Hours) with Cy5 (red). Cy5 labeled DNA from each time point will then be mixed with an aliquot of
the Cy3-labeled “reference” cDNA. In this experimental design, the relative fluorescence intensity
measured for the Cy3 and Cy5 fluors at each array element provides a reliable measure of the
relative abundance of the corresponding mRNA in the two cell populations (i.e. initial vs. later
time points). When there are equal amounts of mRNA present in two cell populations, equal
amounts of Cy3 and Cy5 will bind to the microarray producing a ‘yellow’ image. If some genes are
induced over the time course (higher mRNA levels), then those spots will appear red in the
microarray. Conversely, if a gene is repressed (lower mRNA level) the spot will appear green on
the microarray. In this way, we can monitor all 6000 genes in yeast on a single microarray slide.
The 3DNA Array 350 kit is easy to use and is designed for use with microarrays like ours. The
first step is to reverse transcribe the RNA using a deoxynucleotide triphosphate mix (dATP,
dCTP, dGTP & dTTP), Reverse Transcriptase and special RT dT primers. (Why don’t we just
label the RNA instead?) The dT primer (i.e. poly T) binds to the poly A tail of mRNAs via its 3’
end, the reverse transcriptase enzyme binds to this double stranded region and transcribes a
cDNA. The primers also have special ‘capture’ sequences that are used to bind to fluorescent
dyes on their 5’ ends, (either Cy3 or Cy5). The dyes are attached to the primers after the cDNAs
are synthesized. Many other kits use fluorescently tagged nucleotides, which can result in less
uniformly labeled cDNAs because of sequence dependent incorporation (some sequences are
transcribed more or less efficiently, leading to a bias in the data). The ‘capture’ sequences of the
3DNA primers avoids the problem of sequence dependent incorporation of the dyes. Finally,
we’ll hybridize the cDNA and the fluorescent 3DNA reagents to the microarray in succession. The
fluorescent 3DNA reagent will hybridize to the cDNA because it includes a “capture sequence”
Cell & Molecular Biology Laboratory
BIOL 406
cDNA Probe generation for Yeast Microarrays
2
that is complementary to a sequence on the 5’ end of the RT primer. The signal from these
special primers does not depend on the length or the base composition of the transcript, each
cDNA is labeled by one 3DNA primer.
RNA is extremely sensitive to degradation by RNases.
How carefully you handle your samples and transfer solutions will have a huge
impact on the quality of your microarray data.
Clear your bench of all but the bare essentials.
Wear gloves (at all times!!) and wash down your entire area & pipettors with
'RNase ZAP' (special detergent that helps control RNases a bit.)
All microfuge tubes and pipet tips have only been touched with gloves and have
been sterilized extensively.
Cell & Molecular Biology Laboratory
BIOL 406
cDNA Probe generation for Yeast Microarrays
3
Materials:
(3DNA Array 350 kit Genisphere)
Vial 2
1.0 pmole/µl RT Primer for Cy3 / Alexa Fluor 546 (red cap) or Cy5 / Alexa Fluor 647 (blue cap)
Primer Sequence Information:
Cy3/Alexa Fluor 546: 5' - TTC TCG TGT TCC GTT TGT ACT CTA AGG TGG A –T(17)- 3’
Cy5/Alexa Fluor 647: 5’ - ATT GCC TTG TAA GCG ATG TGA TTC TAT TGG A – T(17)-3’
Vial 3
Deoxynucleotide Triphosphate Mix (10mM each dATP, dCTP, dGTP, and dTTP)
Vial 4
Superase-In™ RNase Inhibitor
Vial 10
Nuclease Free Water
Store Vials 1-12 at –20°C in the dark.
Instructions for Handling Kit Contents
Vials 2
Vials 3 & 4
: Thaw at room temperature, vortex, and briefly microfuge.
: Thaw on ice, briefly microfuge if necessary, and keep on ice at all times. Do not vortex.
Additional Materials:
• RNA sample- (Total RNA sample greater than or equal to 500ng/µl)
• Reverse Transcriptase enzyme - Genisphere RT Enzyme (Genisphere Cat No.
• Reagent Grade Deionized Water (Recommended: VWR Cat No. RC9150-5)
•
RT300320)
0.5M NaOH/50 mM EDTA (cDNA synthesis stop solution)
• 1M Tris-HCl, pH 7.5
• 10 mM Tris-HCI, pH
8.0 / 1 mM EDTA (1X TE Buffer)
• Millipore
Microcon YM-30 Centrifugal Filter Device (30,000 molecular weight cutoff, Millipore
Cat. No. 42409)
Vortex Mixers
80ºC Heating Blocks
42ºC & 65ºC Water Bath Incubators
Microfuge 4ºC
Microfuges RT
NOTES:
Lab bench and pipettors
Before working with RNA, it is always a good idea to clean the lab bench and pipettors with an
RNase decontamination solution (e.g. Ambion RNase Zap Solution).
Gloves and RNase-free technique
• Wear laboratory gloves at all times during this procedure and change them frequently. They will
protect you from the reagents, and they will protect the RNA from nucleases that are present on
skin. • Use RNase-free tips to handle the wash solutions and the Elution Solution, and NEVER
put used tips into the kit reagents.
Cell & Molecular Biology Laboratory
BIOL 406
cDNA Probe generation for Yeast Microarrays
4
Keep reagents on ice
Always close pipet tip boxes
Dispose of tips in waste containers, not on the bench
Never use tips more than once!
Step 1: Preparation of cDNA from Total RNA (Reverse Transcription – RT)
Since microarrays and RNA preparations vary in quality, the exact amount of RNA required for an
experiment will typically range from 1-5 µg of Yeast total RNA.
(10 ul of our samples is approximately 5 µg of Total RNA.)
1. In a microtube, prepare the RNA-RT primer mix:
10µl total RNA (5µg Yeast total RNA)
1µl RT primer Vial 2, 1pmole/µl, either Cy3 (red cap) or Cy5 (blue cap) Fluor
(only use one, NOT both)
- groups assigned to Time Point 1 (9Hrs) should use Cy3 (Reference Time point),
- all other groups (Time Points 13,17 or 21 Hours) should use Cy5
2. Mix the RNA-RT primer mix and microfuge briefly to collect contents in the bottom of the tube.
3. Heat primer mix at 80°C for 10 minutes and immediately transfer to ice for 2-3 minutes.
4. In a separate microtube on ice, prepare a reaction mix for your RT reaction:
4 µl 5X Reverse Transcriptase Buffer
2 µl 0.1M Nuclease Free Water (Vial 10))
1 µl dNTP mix (Vial 3)
1 µl Superase-In™ RNase inhibitor (Vial 4)
1 µl Reverse Transcriptase enzyme, (200 units)
5. Gently mix (do not vortex) the reaction mix and microfuge briefly to collect reaction mix
contents in the bottom of the tube. Keep on ice until used.
6. Add the 9µl of reaction mix from step 5 to the 11µl of RNA-RT primer mix from step 3 (20µl
volume).
7. Gently mix (do not vortex) and incubate at 42°C for 2 hours.
8. Stop the reaction by adding 3.5µl of 0.5M NaOH/50mM EDTA.
9. Incubate at 65°C for 15 minutes to denature the DNA/RNA hybrids and degrade the RNA.
10. Neutralize the reaction with 5µl of 1M Tris-HCl, pH 7.5 and microfuge briefly to collect
reaction mix contents in the bottom of the tube.
11. For Dual Channel Assays (i.e. using two fluorescent dyes):
Groups with time points 13,17 or 21 hours should combine 9 ul of their labeled Cy5 cDNA
with 9 µl of the “Reference” Cy3 DNA (from the Time point 1 group) in a new tube.
Cell & Molecular Biology Laboratory
BIOL 406
cDNA Probe generation for Yeast Microarrays
5
Add 22 ul of 1X TE Buffer to your Cy3/Cy5 “mixed” cDNA (Final Volume 50 µl)
Proceed to Step 2: Concentration of cDNA.
Step 2: Concentration of cDNA
The cDNA must be concentrated before it can be used in the hybridization mix. Although ethanol
precipitation is a traditional method for nucleic acid concentration, this method may lead to
variable results due to partial or complete loss of the pelleted cDNA or incomplete resolubilization of the precipitated cDNA. Microcon concentration is an alternative method that may
offer better performance characteristics.
Note: Evaporative drying of the cDNA is NOT recommended, as a dried sample may result in
lower signal and/or higher background on the array.
Millipore Microcon YM-30 Centrifugal Filter Devices
cDNA samples may be concentrated using the Millipore Microcon YM-30 Centrifugal Filter
Devices (30,000 molecular weight cutoff, Millipore cat. no. 42409). These devices are capable of
reducing the volume of the cDNA synthesis reaction to 3-10µl in as little as 8-10 minutes. The
procedure below reiterates the manufacturer’s directions with minor adaptations for the 3DNA
Array 350 Kit.
Important: When using the Microcon YM-30 column, evaluate centrifuge time and speed to yield
final volumes of 3-10µl. A fixed angle rotor tabletop centrifuge (microfuge) capable of 10-14,000g
should be used.
1. Place the Microcon YM-30 sample reservoir into the 1.5mL collection tube.
2. Pre-wash the reservoir membrane by adding 100µl 1X TE buffer to the sample reservoir. Do
not touch the membrane with the pipet tip.
3. Secure the tube cap and centrifuge for 3 minutes at 14,000g.
4. Add the cDNA to the sample reservoir. Do not touch the membrane with the pipet tip.
5. Secure the tube cap and centrifuge for 8-10 minutes at 14,000g.
6. Carefully separate the sample reservoir from the collection tube. Discard the collection tube.
7. Add 5µl of 1X TE buffer to the sample reservoir without touching the membrane. Gently tap the
side of the reservoir to distribute the TE across the membrane surface.
8. Carefully place the sample reservoir upside down in a new collection tube.
9. Centrifuge for 0.5-2 minutes at top speed in the same centrifuge.
10. Separate the sample reservoir from the collection tube and discard the reservoir. Note the
volume collected in the bottom of the tube (3-10µl). The cDNA sample may be stored for later
use.
11. Add Nuclease Free Water (Vial 10), if necessary, to a final volume of 10µl.
Cell & Molecular Biology Laboratory
BIOL 406
cDNA Probe generation for Yeast Microarrays
THIS is your labeled cDNA which will hybridize to the microarrays next week.
Your sample will be stored at -80ºC til then.
6
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