Lab 6: Quantitative PCR, DNA extraction, epigenetics

Objectives:

• Measure genome-wide methylation using a dot blot.
• Perform qPCR to measure gene expression

Cytosine Methylation Dot Blot

Procedure Background

• There are three primary components to this procedure: making dilutions of your DNA sample, binding your DNA sample dilutions to a nylon membranes (Dot Blotting), chromogenic Immunodetection of methylated cytosines using the Western Breeze kit and a 5meC antibody
• DNA is applied directly on a membrane as a dot using a vacuum manifold. This is then followed by detection of methylated cytosines using a 5-MeC antibody (Antibody product info). For dot blotting we will be using a protocol adapted from Schleicher and Schuel and the Western Breeze manufacture’s protocol for chromogenic innumodetection.

Chromogenic innumodetection consists of the following primary steps:

• Blocking- The membrane is blocked in order to reduce non-specific molecule interactions between the membrane and the antibody. This is achieved by placing the membrane in a solution of non-specific proteins (usually BSA or non-fat milk). The proteins in the blocking solution coat the remaining areas of the membrane where no protein is bound from the transfer. The reason this is necessary is described in the next step.
• Primary Antibody hybridization- The membrane is incubated with the first antibody. The primary antibody is specific for the molecule of interest, which in this case is methylated cytosines, and at appropriate concentrations, should not bind any of the other molecules on the membrane. Remember, antibodies are molecules, too. If we had not blocked the membrane, the antibody would end up binding to both the membrane and your target molecule. This would result in extremely high background (signals not related to the intended target protein(s)) and would use up a significant amount of the available antibody, making the interpretion of results difficult, if not impossible.
• Secondary Antibody hybridization- After rinsing the membrane to remove unbound primary antibody, a secondary antibody is incubated with the membrane. It binds to a species-specific portion of the primary antibody. Due to its targeting properties, the secondary antibody tends to be referred to as "anti-mouse," "anti-goat," etc., depending on the animal species that the primary antibody was created in. This secondary antibody is typically linked to an enzyme that allows for visual identification. In our case, the antibody is linked to an alkaline phosphatase (AP).
• Developing-The unbound secondary antibodies are washed away, and the enzyme substrate is incubated with the membrane so that the positions of membrane-bound secondary antibodies will either change color or emit light. Band densities in different lanes can be compared providing information on relative abundance of the protein of interest. The kit we will be using for visualization (Invitrogen's WesternBreeze Chromogenic Kit) utilizes an enzymatic reaction that creates a dark purple precipitate that can be seen with the naked eye.
• The chromogenic system emplyed in the WesternBreeze Chromogenic Kit is the combination of BCIP (5-Bromo-4-Chlo ro-3'-Indolypho sphate p-Toluidine Salt) and NBT (Nitro-Blue Tetrazolium Chloride). Together they yield an intense, insoluble black-purple precipitate when reacted with Alkaline Phosphatase.

METHYLATED CYTOSINE DOT BLOT PROCEDURE

Supplies and Equipment

• DNA
• 1.5 mL microcentrifuge tubes
• lab pens
• lab coats
• kimwipes
• safety glasses
• gloves
• vacuum manifold
• nylon membrane
• 6X SSC
• filter paper
• hot plate
• large beaker
• floaties for 1.5 mL tubes
• ice buckets
• ice
• microcentrifuge
• micropipettes
• sterile tips for micropipettes
• denaturation buffer
• scissors
• plastic wrap
• UV transilluminator
• Ultra filtered water (Nanopure)
• Blocker/Diluent Part A
• Blocker/Diluent Part B
• plastic dish with cover
• rotary shaker
• blocking solution
• 5-MeC antibody
• TBS-T
• secondary antibody solution
• chromogenic substrate

DNA DILUTIONS

1. If you do not have your own DNA sample, borrow one from a lab mate and note the type of DNA you have chosen
2. Make an initial dilution of your DNA so that you have 50 ng/µL in a total volume of 40 µL
3. Label five snap cap 1.5 ml tubes with your initials, “DNA”, and the appropriate target concentration
4. Prepare five dilutions of your DNA, one of each target concentration, using the table provided. You should have 200ul of each dilution.
5. Set DNA aside to observe set-up dot blot vacuum manifold

Dilution	TARGET amount	ul of H20	ul of 20X SSC	ul of 50ng/ul DNA sample
1	800 ng	124	60	16
2	400 ng	132	60	8
3	200 ng	136	60	4
4	100 ng	138	60	2
5	50 ng	139	60	1

DOT BLOTTING

1. Cut nylon membrane to fit 72 wells of manifold
2. Soak nylon membrane in 6X SSC (enough to cover) for 10 min in top of tip box
3. Cut filter paper to size of the nylon membrane and wet in 6X SSC
4. Assemble manifold with the membrane lying on top of the filter paper
5. Denature DNA in boiling water for 10 m. Immediately transfer to ice
6. Switch on vacuum. Apply 500ul of 6X SSC to each well and allow SSC to filter through. Adjust vacuum speed so that it takes a couple of minutes for the SSC to filter through.
7. Spin down DNA for 5 min
8. Apply entire volume of DNA to wells. Be carful not to touch the membrane.
9. Note where you have applied your samples and each samples concentration on the blot map
10. Allow samples to filter through. If your sample is not filtering, carefully pipette it up and down without touching the membrane.
11. While samples are being pulled through nylon membrane, soak filter paper cut to size in denaturation buffer
12. Once filtered through, dismantle manifold and transfer membrane (dot side up) to filter paper soaked in denaturation buffer and let sit for 5m
13. Place membranes on dry filter paper and let dry
14. Wrap dryed blot in plastic wrap and place DNA-side-down on UV transluminator for 2 mins at 120kJ. This immobilizes the DNA

WesternBreeze® CHROMOGENIC IMMUNODETECTION

General Guidelines

• Avoid touching the working surface of the membrane, even with gloves. Use forceps
• Work quickly when changing solutions as membranes dry quickly.
• Add solutions to the trays slowly, at the membrane edge, to avoid bubbles forming under the membrane. Decant from the same corner of the dish to ensure complete removal of previous solutions.

1. Prepare 20 mL of Blocking Solution

• a. Ultra filtered Water 14 ml
• b. Blocker/Diluent (Part A) 4 ml
• c. Blocker/Diluent (Part B) 2 ml
• d. Total Volume 20 ml

2. Place the membrane in 10 ml of Blocking Solution in a covered, plastic dish.
3. Incubate for 30 minutes on a rotary shaker set at 1 revolution/sec.
4. Decant the Blocking Solution.
5. Rinse the membrane with 20 ml of water for 5 minutes, then decant. Repeat.
6. Prepare 10 mL of Primary Antibody Solution (1:5000 dilution)

• a. Blocking Solution 10 ml
• b. 5-MeC antibody 2 µl
• c. Total Volume 10 ml

7. Incubate the membrane with 10 ml of Primary Antibody Solution for 1 hour. PREPARE YOUR QPCR DURING THIS INCUBATION.
8. Decant primary antibody and wash the membrane for 5 mins with 20 ml of 1x TBS-T. Decant and repeat three more times
9. Incubate membrane in 10 ml of secondary antibody solution for 30 mins. Decant
10. Wash the membrane for 5 mins with 20 ml of TBS-T. Decant and repeat three more times
11. Rinse the membrane with 20 ml of water for 2 minutes, then decant. Repeat twice.
12. Incubate the membrane in 5 ml of Chromogenic Substrate until color begins to develop (1-60 mins)
13. Rinse the membrane with 20 ml of water for 2 minutes, then decant. Repeat twice.
14. Dry membrane of clean piece of filter paper

Quantitative PCR

Supplies and Equipment:

• PCR Plates (white); optically clear caps
• 1.5 ml microfuge tubes (RNAse free)
• Nuclease Free water
• filter tips
• Opticon thermal cycler
• kim wipes
• 2x Immomix Master Mix
• SYTO-13 Dye
• microfuge tube racks
• ice buckets
• timers
• cDNA samples (student provided)

Procedure Background

• Real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR/qrt-PCR) or kinetic polymerase chain reaction (KPCR), is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific sequences in a DNA sample.

• The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real time, a new approach compared to standard PCR, where the product of the reaction is detected at its end. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target.

• Frequently, real-time PCR is combined with reverse transcription to quantify messenger RNA and Non-coding RNA in cells or tissues.

qPCR PROCEDURE

You will run each template (cDNA) in duplicate in addition to two negative controls (no template) - calculate how many reactions this will be!

1. Prepare master mix: Prepare enough master mix for your number of reactions +1 to ensure sufficient volume recovery.

For a 25μl reaction volume:

Component	Volume	Final Conc.
Master Mix, 2X (Immomix)	12.5µL	1x
Syto-13 dye (50uM)	1µL	2µM
upstream primer, 10μM	1.25μl	2.5μM
downstream primer, 10μM	1.25μl	2.5μM
Ultra Pure Water	7uL	NA

2. Add mastermix to wells of a white PCR plate
3. Thaw cDNA samples.
4. Add 2uL cDNA template to each reaction.
5. Add 2uL of ultra pure water to the negative control wells.
6. Cap the wells securely.
7. If necessary, spin the strips to collect volume in the bottom of the wells.
8. Ensure the lids are clean and place strips on ice. (I like to wipe the lids with a clean kimwipe)
9. Load the plate, verify the PCR conditions and start the run (this will be done by your TA).

PCR conditions:
1. 95°C for 10 minutes
2. 95°C for 15s
3. 55 °C for 15 s
4. 72°C for 30 s (+ plate read)
5. Return to step 2 39 more times
6. 95°C for 10s
7. Melt curve from 65°C to 95°C, at 0.5°C for 5s (+plate read)