FACS Procedure - MIT



BEH.109: Laboratory Fundamentals in Biological Engineering.

1o Instructor Prof. Bevin Engelward

MODULE 3

Eukaryotic Cells as Phenotypic Indicators:

The use of RNAi to modulate gene expression

Instructor: Leona D. Samson

Teaching Assistants: Jenn Cheng, Lisa Smeester, Lisa Joslin

Overview of Module 3 Activities

|Monday March 31 |Tues April 1 |Wed April 2 |Thurs April 3 |

|DAY 1 |DAY 1 |DAY 2 |DAY 2 |

| | | | |

|Module 3 Overview & mini-lecture|Module 3 Overview & mini-lecture|Comprehensive lecture on RNAi |Comprehensive lecture on RNAi |

|on RNAi |on RNAi |with some examples |with some examples |

| | | | |

|Safety Orientation |Safety Orientation | | |

| | | | |

|Sterile Technique |Sterile Technique |Harvest transfected cells |Harvest transfected cells |

| | |Microscope analysis & FACS |Microscope analysis & FACS |

|Transfection of EGFP & p53 siRNA|Transfection of EGFP & p53 siRNA|analysis |analysis |

|into EGFP expressing HeLa cells |into EGFP expressing HeLa cells |Analyze data |Analyze data |

|Monday April 7 |Tues April 8 |Wed April 9 |Thurs April 10 |

|DAY 3 |DAY 3 |DAY 4 |DAY 4 |

| | | | |

|Introduction to the ATM, ATR, |Introduction to the ATM, ATR, |Introduction to DNA microarrays |Introduction to DNA microarrays |

|EXO1 and AAG genes |EXO1 and AAG genes |and overview of what will happen|and overview of what will happen|

| | |on days 5 & 6 |on days 5 & 6 |

|Ambion and Blast session to |Ambion and Blast session to | | |

|design new siRNAs for four |design siRNAs for four genes. |Transfect four new siRNAs; |Transfect four new siRNAs; |

|genes. | |cellular RNA will be isolated |cellular RNA will be isolated |

| |siRNA is ordered for next |over the w/e |over the w/e |

|siRNA is ordered for next |experiment | | |

|experiment | | | |

| | |Informal Presentation of FACS |Informal Presentation of FACS |

| | |data by students |data by students |

|Monday April 14 |Tues April 15 |Wed April 16 |Thurs April 17 |

|DAY 5 |DAY 5 |DAY 6 |DAY 6 |

| | | | |

|Label isolated RNA and hybridize|Label isolated RNA and hybridize|Scan microarray slides and |Scan microarray slides and |

|to microarray slides |to microarray slides |analyze results |analyze results |

| | | | |

| | |Wed April 23 |Thurs April 24 |

| | |DAY 7 |DAY 7 |

| | | | |

|Patriots Day |MIT Holiday |MODULE 3 Student Presentations |MODULE 3 Student Presentations |

| | | | |

Sterile Techniques

You will be handling cultured human cells (HeLa cells) during the first two weeks of Module 3 and the following guidelines will help you to prevent your cell cultures from becoming contaminated with bacteria, yeast, moulds and any other unwanted contaminations. PLEASE READ THROUGH THESE GUIDELINES BEFORE BEGINNING YOUR EXPERIMENTS WITH CULTURED HUMAN CELLS.

Hood Preparation

1) Wear gloves to protect yourself but also to prevent dry skin and micro-organisms from contaminating your samples.

2) Swab down the work surface liberally with 70% ethanol. Start from the back and proceed forward. Swab during work if necessary.

3) Swab any instruments that will be used in the hood with 70% ethanol, particularly the pipettes, which will often be used above biological samples.

4) Keep sterile pipette tips in “Hood Only” boxes that are opened only in a sterile environment. Swab the exterior of the box with 70% ethanol.

5) Bottles should always be tightly capped when outside the hood (i.e., they should have been tightly capped the last time they were in the hood).

6) Dry bottles thoroughly if they have been taken out of the water incubator. Swab them with 70% ethanol, especially at the neck and the bottom, and place them directly into the hood. Avoid shaking them vigorously during handling.

7) Bring only the items you need for a particular procedure into the hood to prevent cluttering your working space. Having a clear working space will significantly reduce the chance of contamination! Ensure easy access to items in the hood and maintain plenty of clear space in the center of the hood to work in.

Sterile Handling

1) Spray gloves with 70% ethanol as often as necessary.

2) The indicator stripes on the autoclave tape should turn black if an object has been properly autoclaved.

3) Never block the negative pressure zone (also the frontal non-sterile area) of the vertical laminar flow hood with objects (i.e., notebooks, pipetteman handle).

4) Avoid working too closely to the front, or the non-sterile area, of the hood. Keep working area at the center or towards the back. Keep the objects needed for the current procedure within reach; keep the others in the back.

5) Avoid working above an open bottle or dish in vertical laminar flow. Always work around them unless they are capped or covered.

6) Avoid leaving bottles, dishes, and flasks open when they are not in use. If the cap must be laid down, place it face-up/face-down towards the back of the hood where there is less traffic and less chance of being touched or crossed over. Correct cap placement has been debated. Having a cap facing up can potentially introduce airborne particles and drive non-sterile lid liquid onto the interior face of the cap, where contaminations can fall into the bottle upon recapping. If face-down placement is preferred, then make sure to swab the area specifically and thoroughly before the cap is placed down there. Conversely, if hood surface sterility cannot be absolutely guaranteed due to high traffic or cluttering, then face-up is a better option. The best placement, however, is to place the cap on its side and towards the back of the hood. This way the interior is not in contact with the air flow or with the work surface. However, this is not possible with dishes. Therefore, exercise good judgment in light of individual operating style and the hood setup.

7) Never pour from one sterile container to another. Pouring will generate a liquid path to introduce infection from the outside to the inside. Always pipette or use filters when transferring from one bottle to another.

8) Mop up any spillage immediately and swab with 70% ethanol to avoid the chance of growth of micro-organisms.

9) Withdraw a pipette from its wrappers at the center of the working area, tilt it such that the tip (bottom end) is pointing away from the frontal non-sterile area and away from other objects in the hood.

10) Withdraw the pipette such that it slides through the sterile interior of the wrapper without touching the outside of the wrapper.

11) Avoid contact between the tip of the pipette and the mouth of the bottle. The mouth and neck of the bottle (both inside and out) present a potential source of contamination.

12) When working with Pasteur pipettes, do not remove pipettes directly from the box with fingers. Shake the tube gently to cause the pipettes to slide out slightly, and then withdraw a pipette with fingers without touching the other pipettes and the tube interior.

13) To keep the hood from being cluttered, do not leave any trash in the hood. Immediately discard uncontaminated wrappers in the regular trash. Put all pipette tips and biologically contaminated sharps in the sharps biohazard waste container. Put all biologically contaminated tissue culture plates, flasks, and other non-sharps in the non-sharps biohazard waste container. However, an effort to minimize entry/exit from the hood should be made to minimize disturbances in the laminar flow at the entrance, which may create the potential to waft in contaminants.

14) Handle the pipette with a steady hand. Avoid large motions and do not let the tip touch anything non-sterile. Keep the tip away from the front and far above the objects in the hood.

15) Do not fill a dish/flask so full or swirl it such that the medium spills over the edge. This will introduce a path of infection via liquid and may cause cross-contamination.

Cleaning up

1) Cap bottles tightly before removing them from the hood.

2) Swap down the work surface liberally with 70% ethanol.

3) Turn off the vacuum, if used.

MODULE 3 – Day 1

Transfection of EGFP and p53 siRNAs into HeLa cells

MOST STUDENTS WILL WORK IN GROUPS OF TWO

We will be studying RNAi knockdown of enhanced green fluorescent protein (EGFP) expression in EGFP-expressing HeLa cells. We will transfect EGFP-HeLa cells with duplexed siRNA using a modified oligofectamine transfection protocol.

You can look over the Oligofectamine Transfection protocol provided by Invitrogen at a later date to understand how the conditions are optimized (see below).

Each group will perform three oligofectamine transfections:

1) with EGFP siRNA

2) with p53 siRNA

3) without siRNA.

The p53 siRNA controls for nonspecific siRNA effects, and the experiment without siRNA controls for oligfectamine effects.

The day prior to transfection, cells will be prepared for you at a density of 0.5 to 1 X 105 cells/well in each well of a 6 well plate.

Required Materials:

One 6-well plate of HeLa cells (per 2 students)

30 ml Phosphate Buffered Saline (PBS)

10 ml RPMI media, serum free, antibiotics free

6 eppendorf tubes

On ice in eppendorf tubes:

30 μl oligofectamine

10 μl EGFP siRNA, 33 μM

10 μl p53 siRNA, 33 μM

Transfection Procedure (use sterile technique + read each task entirely before starting).

**NOTE: read through the entire protocol first. You may want to divide the tasks by having one partner start on step 4 while the other partner prepares the reagents in steps 2 and 3.**

1. Label your plates

You will use 2 wells for EGFP knockdown, 2 wells for p53 knockdown, and 2 wells for oligofectamine-only controls. Label your plate accordingly. See example below:

[pic]

2. Label 6 eppendorfs as follows: AGFP, BGFP, Ap53, Bp53, Actrl and Bctrl.

3. Prepare reagents in labeled eppendorfs according to the chart below.

• FIRST pipette the RPMI media (which has no serum or antibiotics) into all tubes.

• THEN pipette the siRNA (EGFP for the EGFP tubes, p53 for the p53 tubes) and oligofectamine needed for each tube. Make sure that you pipette directly into the media and fully depress the pipette.

• Mix each tube by gently tapping on the side.

| |Tube A |Tube B |

| |Media |siRNA |Media |Oligofectamine |

|EGFP |94 μl |6 μl |94 μl |6 μl |

|p53 |94 μl |6 μl |94 μl |6 μl |

|Ctrl |100 μl |------- |94 μl |6 μl |

Transfer entire contents (100μl) of TUBE A into TUBE B for both siRNA transfections, and for the Oligofectamine control. Mix by gently tapping. Incubate at room temperature for 20 min.

4. Wash and prepare the cells while the transfection reagents are incubating:

a. Aspirate off the media.

b. Add 2ml of PBS to each well (add slowly, do NOT forcefully pipette in)

c. Rock plate gently and then aspirate off the PBS.

d. Repeat steps b and c.

e. Add 900ul media to each well

5. After the 20 min incubation, add 95 μl of transfection reagent from the appropriate eppendorf tube (GFP, p53, or ctrl) to each well. Add reagents DROPWISE, close to the surface of the media, and spreading them evenly over each well (see diagram below for even-distribution method). Mix gently by rocking your plate back and forth.

6. Put your plate in the 37°C incubator.

After 6 hours of transfection, the following steps will be performed for you to complete the procedure:

• Add 1ml of RPMI media + serum to each well.

• Incubate overnight @ 37°C, 5% CO2.

• Aspirate media and transfection reagents off. Replace with 1ml RPMI media + serum.

• Return to incubator.

48 hours post-transfection, you will assay the plates by flow cytometry.

[pic][pic]

MODULE 3 – Day 2

Harvest HeLa cells previously transfected with EGFP and p53 siRNAs; analyze EGFP expression by microscopy and flow cytometry

These instructions should look familiar! Note that HeLa cells may be harder to trypsinize and harvest than the embryonic stem cells you worked with in Module 2.

Required Materials:

1 six well plate of HeLa cells from Day 1.

4 ml Trypsin

15 ml serum containing RPMI media

10 ml PBS

5 ml PBS on ice

6 15 ml conical tubes

6 12x75 mm polystyrene flow cytometry tubes

Electric pipettor w/10ml pipettes

P1000 w/tips

Ice

Procedure in lab (read each task entirely before starting):

❑ Visually inspect each of the six well plates under the fluorescent microscope to determine whether the expression of EGFP is altered in any of the wells. Take digital pictures of a typical field of cells for each of the six wells.

❑ Label six 15 ml conical tubes and six flow cytometry tubes, one of each kind of tube per well. An example of how these could be labeled is as follows: EGFP(1), EGFP (2), p53(1), p53(2), Ctrl (1), Ctrl(2).

❑ Pipette off all media from each well into waste (~1 ml per well). Rinse cells by doing the following: Add 1 ml of PBS to each well (pipette slowly into wells, do NOT pipette forcefully), gently rock plate to rinse wells, and then pipette off PBS into waste.

❑ Detach cells by doing the following: Add 0.5 ml of trypsin per well. Put plate in 37(C incubator for 5 min.

❑ After 5 min incubation time, look under microscope to see if cells have detached. If they have not detached, gently knock plates on sides and put back in incubator for 1-2 minutes.

❑ Add 2 ml of media to each well to deactivate the trypsin; add the medium vigorously so that cells become detached. Do not let cells sit in trypsin for longer than necessary. For one well at a time, pipette the cells up and down 5-10 times in order to wash cells off the dish and to break cell-cell contacts and mix cells well. ***Use a P1000 pipettor and make sure to pipette up and down vigorously in the edges of the well.*** Transfer the media plus cells (total volume) to the appropriately labeled 15 ml conical tube.

❑ Pellet the 2.5 ml cell suspension by spinning for 5 min @ 1500 rpm in the 15 ml conical tubes. Make sure lids are seated properly and shut tightly before starting the centrifuge. Since we only have one centrifuge, please make sure that at least two groups are pelleting at the same time.

❑ After spinning down cells, pipette off the media, being careful not to dislodge the cell pellet.

❑ Disperse the cell pellet by flicking the tube. Resuspend the pellet in 300ul ice cold PBS. Pipette sample up and down to resuspend cells, and then transfer immediately to polystyrene flow cytometry tubes and put cells on ice. You are ready to go to the FACS center.

Flow Cytometry Analysis (for more info: ):

1. Turn the Flow Cytometer on. Turn the computer on.

2. Get user name and password from TA.

3. Get an acquisition file from TA.

4. Follow instructions on FACS machine.

5. Points to remember for FACS:

a. During the setup:

1. Make sure to press the “Set” button after selecting the data file in the instrument settings menu.

2. Set the analyzer to count 20,000 cells for each sample.

b. Run the Negative Control samples first.

c. Be sure to flush with water by fitting the water tube over sipper after every sample.

d. Print a copy of each sample run.

6. Shut down according to the notes on the FACS machine.

MODULE 3 – Day 3

Design siRNAs to knock down the expression of four genes that influence how cells respond upon exposure to DNA damaging agents; ATM, ATR, EXO1 and AAG

Each group will design an siRNA sequence to knock down each of the four human genes listed below. i.e., every group will design siRNAs for all four sequences. Each gene is shown with its full name, common abbreviation, mRNA accession number, and the base pair region to be targeted.

1. Ataxia telangiectasia mutated (ATM)

NM_000051

bp 8700-8800

2. Ataxia telangiectasia and Rad3 related (ATR)

NM_001184

bp 4450-4550

3. 3-alkyladenine-DNA glycosylase (AAG, a.k.a. MPG)

NM_002434

bp 700-800

4. Exonuclease 1 (EXO1)

NM_003686

bp 1500-1600

The design of the siRNA will be based on Tuschl et al.’s criteria ().

These criteria have been incorporated into a search algorithm that was developed by a company called Ambion. Ambion’s website contains a search engine that will find multiple candidate sequences for each gene. You will identify candidate sequences that fall within the given base pair region for each gene. You will then perform BLAST searches to determine which candidate sequence has the highest selectivity for your gene of interest. For your interest the home page of Ambion is shown below.

[pic]

1) Finding the mRNA sequence of the gene target.

a. Go to the NCBI homepage: .

b. Enter the accession number for your gene of interest.

c. Make sure “Nucleotide” is selected from the left hand pull-down menu.

d. Click on the Go button.

e. Click on the link for your gene of interest.

f. Display the mRNA sequence in FASTA format by selecting “FASTA” from the pull-down menu at the upper left next to the display button. Click on the Display button.

2) Finding candidate siRNA sequences.

a. Go to the Ambion siRNA design tool website: .

b. Copy and paste the mRNA sequence for your gene of interest into the mRNA sequence box.

c. Select end my siRNAs with TT.

d. Select “all G/C contents” for G/C content maximum.

e. Click on the Submit button.

3) Selecting an siRNA sequence.

a. Only consider the target sequences that fall within the given base pair range for your gene of interest. (“Position in gene sequence” is listed for each sequence).

b. Eliminate sequences that fall outside the 30-70% G/C content range.

c. BLAST the remaining sequences.

i. Click on the BLAST search link under the target sequence.

ii. In the BLAST window, scroll down to “options for advanced blasting” and limit the search so that you are only screening Homo Sapiens entries by selecting “Homo Sapiens [ORGN]” from the dropdown menu.

iii. You do not need to change any of the options in the “format” section.

iv. Click BLAST.

4) Analyzing BLAST results:

a. The BLAST results show “sequences producing significant alignments”. You want to make sure that all of the sequences showing up as good matches are either your gene of interest or some derivative of your gene of interest.

i. Find all sequences that have a “Score” of 42 bits, corresponding to “Identities = 21/21 (100%)”. The scores can be found in the list of “sequences producing significant alignments”. Scroll down past this list to the “alignments” section for more detailed information about each sequence.

ii. For each one, answer the following questions:

1. Is it your gene of interest? *

2. Is it a transcript variant of your gene of interest?

3. Is it a clone, BAC, or contig of your gene of interest? **

iii. If you answered NO to ALL of the questions above then the target sequence you just BLASTed has significant homology to another gene and cannot be used.

iv. If you answered YES to at least ONE of the above questions then the sequence is a possible candidate for use.

v. HINTS:

* The same gene may go by different names. Look at the original NCBI summary of your gene of interest from step 1 above. Under “features: gene” some synonyms for your gene may be listed.

* If you think it might be the same gene but cannot tell, you can do an alignment. Go to: . Paste the FASTA sequences of your gene of interest (the same mRNA sequence that you used for your search on ambion’s website) and the suspected match into the first and second sequence fields and click “perform align”. Check for a high identity score (>90%).

** To determine if the BAC or clone is from the correct chromosome, you can check the genomic location of your gene. Look at the original NCBI summary of your gene of interest from step 1 above. Click on “Link” in the upper right hand corner, and select “Map Viewer”. The chromosome on which your gene of interest is located should be highlighted in the list of chromosomes at the top. The chromosome from which the BAC or clone was obtained is usually listed in the BAC or clone description, OR use map viewer, as above.

a. After completing the above steps, it is likely that you will still have several candidate target sequences. You will decide which one is best by looking at the next highest matches in the BLAST search for each target sequence.

i. Eliminate sequences that show a 20/20 or 19/19 identity with a gene that is not your gene of interest. Use the three criteria in (4aii) to determine this.

ii. Select the target sequence with the lowest homology to any gene that is not your gene of interest. Compare the next highest matches that are not 21/21, 100% identity matches. Are these matches to your gene of interest? If not, we would like for our target siRNA sequence to have LOW homology to these other genes. For example, siRNA target sequence #1 shows 18/18 (score=36) as the next highest match to a gene that is not our gene of interest. siRNA target sequence #2 shows 17/17 (score=34) as the next highest match to a gene that is not our gene of interest. siRNA #2 would therefore be a better match than siRNA #1, since it has LOWER homology to other genes. Likewise, a target siRNA sequence with 16/17 identity match to a different gene is preferred over one with 17/17 identity match to a different gene, because it has lower homology to other genes.

iii. If you are left with more than one target sequence after eliminating all those with greater homology to other genes, select the target sequence that gives the least number of matches to other genes at the next highest match level after 21/21 (e.g. 15/15).

MODULE 3 – Day 4

Transfection of ATM, ATR, EXO1 and AAG siRNAs into HeLa cells

STUDENTS WILL CONTINUE TO WORK IN GROUPS OF TWO

We will be studying RNAi knockdown of ATM, ATR, Exo1, AAG, EGFP, and p53 expression in EGFP-expressing HeLa cells. We will transfect EGFP-HeLa cells with duplexed siRNA using a modified oligofectamine transfection protocol. This time, we will assay the cells for mRNA expression using DNA microarrays.

Each group will perform two types of oligofectamine transfections:

1) with siRNA (each group will knockdown 1 of the 6 genes)

2) without siRNA.

Again, the experiment without siRNA controls for oligfectamine effects.

The day prior to transfection, cells will be prepared for you at a density of 0.5 to 1 X 105 cells/well in each well of a 6 well plate.

Required Materials:

One 6-well plate of HeLa cells (per 2 students)

30 ml Phosphate Buffered Saline (PBS)

10 ml RPMI media, serum free, antibiotics free

4 eppendorf tubes

On ice in eppendorf tubes:

30 μl oligofectamine

10 μl siRNA, 100 μM (each group will have a different gene)

Transfection Procedure (use sterile technique + read each task entirely before starting).

**NOTE: read through the entire protocol first. You may want to divide the tasks by having one partner start on step 5 while the other partner prepares the reagents in steps 3 and 4.**

1. Label your plates

You will use 4 wells for siRNA knockdown of your gene and 2 wells for oligofectamine-only controls. Label your plate accordingly. See example below given for the group doing ATM:

2. Label 4 eppendorfs as follows: AATM, BATM, Actrl and Bctrl. (Label with the gene that your group is doing)

3. Prepare reagents in labeled eppendorfs according to the chart below.

• FIRST pipette the RPMI media (which has no serum or antibiotics) into all tubes.

• THEN pipette the siRNA and oligofectamine needed for each tube. Make sure that you pipette directly into the media and fully depress the pipette.

• Mix each tube by gently tapping on the side.

| |Tube A |Tube B |

| |Media |siRNA |Media |Oligofectamine |

|ATM |196 μl |4 μl |188 μl |12 μl |

|Ctrl |100 μl |------- |94 μl |6 μl |

4. Wash and prepare the cells while transfection reagents are incubating.

a. Aspirate off the media

b. Add 2ml of PBS to each well (add slowly do NOT forcefully pipette in)

c. Rock plate gently and then aspirate off the PBS.

d. Repeat steps b and c.

e. Add 900ul media to each well

f. Incubate for 20 min

5. After the 20 minute incubation, add 95 μl of transfection reagent from the appropriate eppendorf tube (siRNA or ctrl) to each well. Add reagents DROPWISE, close to the surface of the media, and spreading them evenly over each well (see diagram below for even-distribution method). Mix gently by rocking your plate back and forth.

6. Put your plate in the 37°C incubator.

After 6 hours of transfection, the following steps will be performed for you to complete the procedure:

• Add 1ml of RPMI media + serum to each well.

• Incubate overnight @ 37°C, 5% CO2.

• Aspirate media and transfection reagents off. Replace with 1ml RPMI media + serum.

• Return to incubator.

48 hours post-transfection, the cells will be harvested, the RNA isolated (this will be done for you) and this RNA will used for the DNA microarray analysis on Days 5 & 6.

MODULE 3 – Day 5

Label the mRNA isolated from HeLa cells transfected with siRNAs targeted to the EGFP, p53, ATM, ATR, EXO1 and AAG gene transcripts and hybridize to DNA microarray slides

MOST STUDENTS WILL WORK IN GROUPS OF THREE

The total RNA from your previous transfection experiments has been isolated for you over the weekend. For the two class sections combined, 8 chips total will be hybridized in order to do compare the gene expression profiles of the following 8 sets of cells:

1. EGFP knockdown vs. oligofectamine-only treated cells

2. p53 knockdown vs. oligofectamine-only treated cells

3. ATM knockdown vs. oligofectamine-only treated cells

4. ATR knockdown vs. oligofectamine-only treated cells

5. Exo1 knockdown vs. oligofectamine-only treated cells

6. AAG knockdown vs. oligofectamine-only treated cells

7. oligofectamine-only vs. oligofectamine-only treated cells

8. untreated vs. oligofectamine-only treated cells

Chip # 7 (above) will control for cDNA synthesis and microarray hybridization effects. Chip # 8 will control for effects specific to oligofectamine treatment. Each group is responsible for completing one of the 8 microarray experiments, and the data will be shared with the entire class. Each group will reverse transcribe mRNA from each of their two cell populations, label the cDNA, and hybridize the cDNA to a DNA microarray (one microarray per group).

Before you begin, take note of the following:

• RNase comtamination is everywhere! You must use gloves, and spray your benches with RNase away. Wipe off pipettes with RNase away as well.

• The dyes that are used are light sensitive, and must be shielded from light. Protect them from degradation by wrapping tubes in foil.

• Do not touch the surface of the DNA microarray or the DNA will be wiped off.

Required Materials:

For the cDNA Synthesis (all on ice):

|10 μl total RNA from knockdown or control cells |Cy3 dye |

|10 μl total RNA from oligofectamine treated cells. |Cy5 dye |

|mRNA alien spike 1 |5X 1st Strand Buffer |

|mRNA alien spike 2 |Super Script II Reverse Transcriptase (200U/ml) |

|Master Mix |0.5M EDTA |

|DEPC dH2O |1N NaOH (equivalent to 1M NaOH) |

|0.2M DTT |1 M Tris, pH 7.5 |

|For the Prehybridization: |For the cDNA Clean-up (at room temperature): |

|1 Microarray chip (per group) |cDNA from cDNA synthesis step |

|1 Lifter slip |1 Qiagen column (per group) |

|1 Hybridization chamber |Buffer PB |

|Prehybridization buffer |Buffer PE |

|Filtered dH2O in 50 ml conical tube |dH2O pH7 |

|100% Isopropanol in 50 ml conical tube |1 eppendorf tube |

| | |

|For the Hybridization: | |

|cDNA from clean-up step | |

|1 Hybridization chamber | |

|1 lifter slip | |

|ddH2O | |

|1 Ziploc bag | |

cDNA Synthesis and Hybridization Procedure (read each task entirely before starting).

Preparation

1. Establish “RNA ONLY” benchtop, eppendorf tubes and pipettes.

2. Wipe gloves, pipettes and surfaces down with RNase away before proceeding.

• cDNA Synthesis Part I.

You will begin with your 2 eppendorfs, each containing 10 μl of total RNA, one from the siRNA knockdown, one from the control cells. Add reagents to these two eppendorfs according to the following steps.

1. Add 1 μl Oligod(T)18 to each sample.

2. Add 1 μl mRNA alien spike 1 to each sample.

3. Add 1 μl mRNA alien spike 2 to each sample.

4. Add 2.4 μl DEPC dH2O to each sample.

5. Incubate at 65°C for 10 min.

6. Incubate at 25°C for 5 min.

Prepare for light-sensitive cy dyes by putting foil over your tube rack.

7. Add 4 μl Cy3 dye to knockdown sample (oligofectamine-treated/untreated for 7 & 8).

8. Add 4 μl Cy5 dye to oligofectamine-treated control sample.

9. Incubate at 42°C for 2 min.

10. Add 8.6 μl Master Mix to each sample.

11. Add 2 μl RT Super Script II to each sample.

12. Incubate at 42°C for 60 min.

• Prehybridization of Chip. Remember, do not touch the surface of the DNA microarray or the DNA will be wiped off.

1. Place chip array-side up in hybe chamber with lifter slip on top.

2. Add 30 μl prehybe solution to edge of lifter slip on top of array. The solution should get sucked under the slip and cover the entire array area through capillary action.

3. Incubate at room temp for 45 minutes.

4. Wash slide by placing slide in filtered dH2O in conical tube. Shake gently until lifter slip comes off. Wash for 2 minutes.

5. Wash slide in 100% isopropanol in conical tube for 2 minutes.

6. Air dry slide (wick with kim wipe).

• cDNA Synthesis Part II.

Your 2 eppendorfs, containing mRNA that you reverse transcribed and labeled should be done incubating. Please proceed with the following steps.

1. Cool on ice for 5 min.

2. Add 3 μl 0.5M EDTA to each sample

3. Add 3 μl 1N NaOH to each sample.

4. Incubate at 70°C for 15 min.

5. Cool on ice for 5 min.

6. Add 7.5 μl Tris pH 7.5 to each sample.

• cDNA Clean-up.

You will now purify the cDNA that you synthesized and labeled in the previous steps. Each group will use one QIAgen column.

1. Add 250 μl Buffer PB to each sample.

2. Load the entire contents of both samples (Cy3 labeled knockdown cDNA + Cy5 labeled control cDNA) onto the QIAgen column.

3. Spin at maximum speed in microfuge for 1 minute.

4. Discard eluant (flow through).

5. Add 650 μl Buffer PE to each sample.

6. Spin at maximum speed for 1 minute.

7. Discard eluant.

8. Spin at maximum speed for 1 minute to get rid of all PE.

9. Discard eluant.

10. Transfer the column to a new eppendorf tube.

11. Elute the cDNA by adding 30 μl dH2O pH7 (room temp) directly onto column membrane.

12. Incubate for 2 minutes at room temp.

13. Spin at maximum speed for 1 minute.

14. Discard the column, keeping the eppendorf tube containing your eluted cDNA.

15. Add 30 μl of hybridization buffer to the tube.

• Hybridization.

You will now hybridize the cDNA that you synthesized, labeled and purified in the previous steps, to a DNA microarray. Each group will use one microarray.

1. Place array in Corning hybridization chamber.

2. Place lifter slip on top of array.

3. Denature labeled cDNA by placing tube in 80(C heat block for 2min.

4. Chill on ice for 2min.

5. Spin at maximum speed for 10 seconds.

6. Slowly apply labeled cDNA in hybridization solution to edge of slip. Capillary action will draw solution over the array.

7. Add 10(l ddH2O to both reservoirs in chamber, cover and reassemble hybridization chambers.

8. Place chambers in Ziploc bag(s) and place in 42(C light proof water bath. Weigh down bag(s) with a plastic tube rack.

After 16 hours of hybridization, the microarrays will be washed for you. You will scan them during the next class period.

MODULE 3 – Day 6

Scan DNA microarray slides and analyze the results

STUDENTS WILL CONTINUE TO WORK IN GROUPS OF 3

Each group will have scan the microarray that they hybridized during the previous class. Together as a class, we will analyze the data generated from all 8 microarrays.

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download