Northern Analysis

As the only technique with the capability of simultaneously measuring the size, amount and abundance of RNA molecules, Northern blotting is an important technique in studying eukaryotic gene expression. Northern blotting is based on resolving the RNA molecules according to their size in a MOPS-formaldehyde gel, followed by transferring to a membrane. The blotted membrane will by hybridized by a labelled RNA probe complementary to the target RNA. Detection of the labelled RNA signal by autoradiography or phosphorimaging will determine the size and amount of the target RNA. In this experiment, we will use Northern analysis to study the HSPA1A (a human 70kDa heat-shock protein) transcriptional level upon heat-shock in HeLa cells. Heat-shock proteins are transcriptionally regulated and as intra-cellular chaperons prevent unfolding of proteins in the cells exposed to elevated temperature or other kinds of stress. For Northern blotting, we will use the “QIAGEN RNasey Mini Kit” to extract RNA from HeLa cells, with and without heat-shock.

Outline:

A. Tissue Culture

  1. Heat-shock and harvesting cells

B. Preparation of the blot

  1. Preparation of RNA Samples
  2. Preparation of a 1% Denaturing Agarose Gel
  3. Sample Analysis
  4. Preparation of the Blot

C. Making the Riboprobe

  1. Preparation of the Riboprobe Construct
  2. In vitro Transcription Using alpha-32P CTP

D. Blot Development

  1. Prehybridization of the Blot
  2. Hybridization of the Blot
  3. Blot Development

E. Appendix

  1. Notes for RNA Handling
  2. Anticipated Results
  3. Solutions

A. Tissue Culture

  1. Heat-shock and harvesting cells

    2. ** Steps i-iii have already been performed by Marc Morgan.

    i. Collect HeLa cells in DMEM + 10% FBS and adjust cell concentration to 4×10^6 cells/mL. Make 1 mL aliquot for each sample. For GeneX course eight 10 cm dishes yielded ~ 8×10^7 cells total (enough for 20 samples).

    ii. Put cells into 42C water bath (heat shock) or 37C (control) for 2 hours.

    iii. Collect cells by centrifugation (200xg, 5 min), aspirate medium and lyse in 700 uL RLT (lysis buffer) containing 1% 2-mercaptoethanol. Store at -80C until ready for RNA purification.

B. Preparation of the Blot

  1. Preparation of RNA Samples using Qiagen RNeasy Mini columns

    2. Note: Consider “Notes for RNA Handling” (Appendix) before working with RNA samples.

    ** Make sure ethanol has been added to solution RPE before starting **

    i. Thaw 700 uL RNA lysates in RLT at room temperature.

    ii. Add 700 uL 70% ethanol to the samples and mix thoroughly by inverting.

    iii. Add 700 uL of sample to a Qiagen RNA Mini column. Remember to label the columns so you know which sample is which.

    iv. Spin at max speed 30 sec. Discard flow through.

    v. Repeat steps iii.-iv. for the remaining 700 uL of sample.

    vi. Add 700 uL RW1. Spin, max speed, 30 sec. Discard flow through.

    vii. Add 500 uL RPE. Spin, max speed, 30 sec. Discard flow through.

    viii. Repeat step vii.

    ix. Spin empty column for 2 min to remove any residual RPE buffer.

    x. Transfer column to an RNase free collection tube. Remember to label the collection tubes corresponding to the column labels.

    xi. Add 60 uL (30 uL) RNase free H2O to the column bed. Incubate 1 min at room temperature.

    xii. Spin 1 min at max speed. The eluted RNA is in the flow through.

    xiii. Quantify RNA by Nano-Drop. The concentration should by ~ 1 ug/uL.

    xiv. Remove 2x 10 ug of RNA from each sample for Northern analysis, or store at -80C until ready to run the Northern.

  2. Preparation of a 1% Denaturing Agarose Gel

    3. Each Group will only need 6 lanes of a gel so if there aren’t enough tanks some groups can share gels. Remember to note the order that you load your samples and separate them with markers.

    ** Before starting, treat gel tanks, casting tray and comb with NaOH for > 30 min. Then wash extensively with ddH2O. **

    i. Combine: 95 mL H2O and 1.25 g Agarose.

    ii. Boil in microwave carefully (you don’t want to evaporate too much water).

    iii. Allow the gel to cool until it is no longer very hot to touch.

    iv. IN THE FUME HOOD, Add: 12.5 mL 10x MOPS buffer and 20.7 mL 37% Formaldehyde. Swirl several times to mix thoroughly.

    v. IN THE FUME HOOD, pour gel into the casting apparatus. Be careful not to make the gel too thick (you only need to load about 20 uL of sample).

    vi. Cover the gel with plastic wrap and let it set at least 30-45 min.

  3. Sample Analysis

    4. i. Thaw a 2x 5 uL aliquots of RNA Marker and a 2x 10 ug aliquot of each RNA sample (Control and Heat Shocked) on ice.

    Loading will look like this:
    Lane #1: Markers, Lane #2: 10 ug Control, Lane #3: 10 ug Heat Shock, Lane #4: Markers, Lane #5: 10 ug Control, Lane #6: 10 ug Heat Shock
    There are 2 replicates you will be hybridizing 2 probes: HSPA1A (HSP70) and a Histone H1 loading control.

    ii. Dry down RNA in speedvac (65C for 30 min). Samples don’t need to be completely dry but should just be a small droplet (~ 2 uL).

    iii. Prepare RNA sample buffer:

    500 uL RNA sample buffer
    50 uL 10x MOPS
    50 uL 10x Dye Buffer (50% glycerol, 10 mL EDTA, 0.25% BP Blue)
    250 uL Formamide
    100 uL 37% Formaldehyde
    50 uL H20
    4 uL Gel Red (10,000 stock)

    iv. Add 20 uL (30 uL) RNA sample buffer to RNA.

    v. Heat to 65C for 20 min to denature RNA, then IMMEDIATELY transfer onto ice and let sit for 2 min.

    vi. Perform a quick spin to collect the sample in the bottom of the tube.

    vii. Put the MOPS-Formaldehyde gel into the running tank and fill with 1x MOPS running buffer.

    viii. Load samples and markers onto the gel.

    ix. Run gel at 20V overnight. The following day, finish running at 50V (run until blue is about 1 inch from the bottom).

  4. Preparation of the Blot

    5. i. Take an image of the gel alongside a UV ruler that is adjusted so that the zero is adjacent to the wells of the gel.

    ii. Rinse the gel several times with ddH2O.

    iii. Soak the gel in 20x SSC with rocking for 20 min.

    iv. Prepare the transfer stack (detailed below in step v.-xvi.)

    v. Cut 3 pieces of Whatman paper to 13cm x 30cm piece to be used as the wick. Also cut 3 pieces of Whatman paper tp 13cm x 10cm pieces, these will go on top of the gel and membrane.

    vi. Flip the gel casting tray upside down and put it into a Pyrex dish containing 20x SSC.

    vii. Put the 3 Whatman wicks (13cm x 30cm) on top of the tray so that the long ends sit in the 20x SSC buffer and absorb it. Pour some 20x SSC onto the wick sheets to fully wet them and roll out any air bubbles with a serological pipette (5 mL or 10 mL pipette works well).

    viii. Put a thin tim of parafilm around the very edge of the casting tray, on top of the Whatman wick, to prevent buffer from wicking around the gel. This isn’t absolutely necessary, but it will help get a better transfer.

    ix. Cut the wells off of the gel and flip it over so that the underside of the gel is now facing up. The RNA is closer to the bottom of the gel so this will make the RNA transfer more efficiently. Carefully roll out any bubbles.

    x. Wet a 13cm x 10cm piece of HYBOND-N membrane with ddH2O and then 20x SSC and place it on top of the gel. Carefully roll out any bubbles.

    xi. Place the 3 sheet of 13cm x 10cm Whatman on top of the Hybond-N. Gently toll out any bubbles.

    xii. Cut sheets of paper towel to the same dimensions as the transfer stack and put 2-3 inches worth of paper towels on top of the Whatman. Try to minimize gaps in the paper towel stack since this is going to absorb the buffer as it wicks through the gel.

    xiii. Find a flat object that will cover the top of the stack (i.e. glass plate, lid of large pipette tip box).

    xiv. Place a weight on top of the stack, something that weighs around 500g is optimal.

    xv. Note if your stack seems a bit unstable, anchor it with long strips of tape. The stack will typically shift overnight so you don’t want it to fall over.

    xvi. Transfer overnight by capillary transfer

    Order of transfer stacks (bottom to top):
    Pyrex Dish
    20x SSC
    Casting tray (flip upside down)
    3x Whatman wick (13cm x 30cm)
    Gel (Cut off wells, flip gel over)
    Hybond N (13cm x 10cm, wet in H2O, then 20x SSC)
    3x Whatman (13cm x 10cm)
    Tissue Paper (Kimberly-Clark, Wypall, L40)
    Flat plate
    Weight

    Blot

    xvii. The following day disassemble the stack and put the Hybond-N membrane onto a clean piece of saran wrap.

    xviii. If you can see the stained RNA ladder, mark the bands with a pencil.

    xix. Put the blot onto a UV light box alongside a UV ruler and mark the RNA ladder bands and ribosomal-RNA bands with a pencil. Importantly, mark clearly where you will need to cut the blot.

    xx. Crosslink with two cycles of the UV Stratalinker on the Auto Crosslink setting.

    xxi. Cut the blots and label them. Dry the membrane between pieces of Whatman Paper and wrap in plastic wrap or aluminium foil until it is ready to be hybridized. The membrane can be stored at room temperature.

C. Making the Riboprobe

  1. Preparation of the Riboprobe Construct

    2. Briefly, the aim of this step is to design and prepare a riboprobe complementary to the target RNA. We use T7 phage RNA polymerase for in vitro transcription. First, we clone a cDNA of our gene of interest into a cloning vector that has the T7 RNA polymerase promoter sequence. We want to clone our cDNA in the “antisense” direction, such that T7 RNA polymerase is transcribing a RNA molecule complementary to the RNA in the cell (e.g. a protein coding mRNA). Once our fragment is cloned in the right direction, we will use Promega’s “Riboprobe System – T7” for the in vitro transcription step. In this experiment, using two constructs, we will prepare two different riboprobes. First, a 1926b riboprobe complementary to the HSPA1A mRNA (2445b) to study transcription level of this heat-shock protein in HeLa cells knocked-down for AFF4 and upon heat-shock stress. And the second is a 681b riboprobe for detection of the HIST1H1B mRNA (790b), the level of which is not expected to change significantly by heat-shock, and will be used as a loading control in our experiment.

    For more details see:
    Molecular Cloning: a laboratory manual, Sambrook J. and Russel D. W., Volume 2, section 9.29
    http://www.promega.com/hr/resources/protocols/technical-manuals/0/pgem-t-and-pgem-t-easy-vetor-systems-protocol/
    http://www.promega.com/hr/resources/protocols/technical-manuals/0/riboprobe-in-vitro-transcription-systems-protocol/

    (Plasmid template prep, linearization and purification have already been performed by Marc Morgan at Northwestern University, you can proceed directly to probe the synthesis)

  2. In vitro Transcription Using alpha-^32P CTP

    3. (Do this step in parallel with “Prehybridizing the Blot”)

    i. To make the ratio-labelled RNA probe, combine the reagents in the same order (A-H) with the table below:

    blot_table

    *Combine equal volume of 10 mM of each rATP, rGTP & rUTP plus one volume of ddH2O to make the overall concentration of 2.5 mM.

    ** Make sure to dilute cold rCTP 1:100 to make a 100 uM stock.

    ii. Incubate for 1 hour at 37 C.

    iii. Add 1 uL of RQ1 RNase-Free DNase.

    iv. Incubate at 37C for 15 min.

    v. To stop the reaction, add 2 uL of 10% SDS and 1 uL of 0.5 M EDTA.

    vi. Bring the reaction to a total volume of 50 uL by adding 26 uL of RNase-free water.

    vii. Obtain a Bio-Rad Micro “Bio-spin 30 column” from 4C.

    viii. Invert the column multiple times to evenly resuspend the gel filtration resin.

    ix. Remove the cap and drain the gel by gravity flow into a 2 mL collection tube. Discard the flow-through.

    x. Replace the column in the collection tube and centrifuge at 1000xg for 2 min.

    xi. Place the column in a clean 1.5 mL labelled tube.

    xii. Apply the 50 uL probe sample and centrifuge for 4 min at 1000xg.

    xiii. Test the alpha-^32P rCTP incorporation by comparing column and flow-through tube with the Geiger counter. The large riboprobe should go around the gel filtration matrix because it is too big for the pores of the gel beads, while the small unicorporated nucleotides and other salts will stay within the pores of the beads. Expect both column and flow-through to be very hot.

D. Blot Development

  1. Prehybridizing the Blot

    2. (Do this step in parallel with “In vitro Transcription Using alpha-^32P CTP”)

    i. Heat the NorthernMax Prehybridization/hybridization buffer solution in a 55C water-bath.

    ii. Label each blot with the name of the probe that you will use. Wet blots briefly in 20x SSC and transfer to glass hybridization tubes. Make sure that the RNA side is facing the inside of the tube. Label the outside of the tubes with a piece of tape with the name of the probe.

    Note: Make sure that the hybridization tubes seem intact and have rubber seals in the lids. If you see any leaking during the prehybridization let the instructor know to get you a new tube.

    iii. Add 25 mL of prehybridization solution to each tube.

    iv. Prehybridize in a 65C oven for 1 hour with 4 rpm rotation.

  2. Hybridizing the Blot

    3. i. Remove the Hybridization tubes from the oven and discard the pre-hybridization solution. This can go in the regular waste since it’s not radioactive.

    ii. Add 25 uL of fresh hybridization buffer (pre-warmed to 55C).

    iii. Very carefully add the appropriate radiolabelled probe to each hybridization tube. If possible put the tube into a tube rack so it cannot fall over. If you spill the probe at this step it will be a huge radioactive mess.

    iv. Carefully seal the hybridization tubes. It is NOT necessary to over tighten the tubes, if you aren’t sure if the seal is OK, get the instructor to check your tube.

    v. Hybridize at 65C with 4 rpm rotation overnight.

  3. Developing the Blot

    4. Note: For these steps the hybridization tubes will have a pretty high temperature and may be painful to hold onto. It is advisable to wear double (or even triple) gloves or to use “hot-hands” grips to hold the tubes. Also, before starting prepare a bunch of paper strips to dab up drops of buffer on the mouth of the hybridization tube so that you avoid radioactive contamination.

    i. Pour the hybridization solution into a 32P waste container. This solution contains the majority of the radioactivity, if you spill at this step it will be a huge radioactive mess.

    ii. Add 25-50 mL of Northern wash A to each of the hybridization tubes, tighten the lids and gently invert 3-5 times to rinse the blot. Gently and carefully pour the wash buffer into the 32P waste container.

    iii. Add another 25-50 mL of Northern wash A to the tubes, tighten the caps and incubate in 65C oven for 20 min with 4 rpm rotation. Discard wash solution in the 32P waste container.

    iv. Add 25-50 mL of Northern wash B in each of the hybridization tubes, tighten the caps, and incubate in 65C oven for 20 min with 4 rpm rotation. Discard wash solution in the 32P waste container.

    v. Repeat for two more washes with Northern wash B and discard the wash solutions in the 32P waste container.

    vi. Using forceps, remove the membranes from the tubes and carefully place in plastic wrap, realigning the two membrane pieces back together.

    vii. Place the wrapped membrane in a film cassette and place a Kodak Bio-Max MS film on top.

    viii. Make two exposures, 1 hour and overnight. Mark the film with the location of the size markers.

E. Appendix

  1. Notes for RNA Handling

    2. – Wear 2 clean pair of disposable gloves and change them whenever necessary throughout the process.
    – Prepare all solutions with RNase-free or DEPC-treated water.
    – To inactivate RNase, if possible, bake glassware for 4 hours at 300C. Autoclaving is not sufficient for RNase inactivation.
    – Rinse plastic ware or glassware with 0.2 M NaOH and DEPC-treated water before use. MilliQ or high quality water is also RNAse free.
    – Maintain a complete set of glassware, plastic ware and buffers to be used just for RNA work. Keep covered with plastic wrap to keep clean.
    – Use sterile RNase-free plastic ware, whenever possible.

  2. Anticipated Results

    3. blot_result

  3. Solutions

    4. 10X MOPS for 1 L
    400 mM MOPS – 41.85 g (4-morpholinopropanesulfonic acid, MW = 209.27)
    50 mM Sodium Acetate – 4.1 g (Sodium Acetate, MW = 82.03)
    ddH2O – 800 mL RNase-free H2O-stir ingredients on a stir plate
    10 mM EDTA – 20 mL of DEPC treated 0.5 M Na2EDTA

    Adjust pH to 7.0 with 10 M NaOH or Glacial Acetic Acid
    Adjust volume to 1 L with DEPC treated H2O
    Divide into 200 mL aliquots, wrap in aluminum foil to protect from light, and store at 4 C.

    Note: Straw colored MOPS is still good MOPS, but yellow MOPS is not so good.

    RNA Loading Buffer
    50% Glycerol – 25 mL
    1 mM EDTA, pH 8.0 – 100 uL 0.5 M
    0.4% bromophenol blue – 0.2 g
    ddH2O – up to 50 mL

    20X SSC
    NaCl – 175.3 g
    Tri-Sodium Citrate – 88.2 g
    Adjust pH to 7.0 with citric acid
    ddH2O – up to 1 L
    Filter to sterilize

    Denhardt’s Solution
    Ficoll 400 – 2 g
    Polyvinilpyroldone – 2 g
    BSA – 2 g
    ddH2O – up to 1 L

    Hybridization Solution for 500 mL
    50% formamide – 250 mL
    3.5% SDS – 17.5 g
    150 mM NaPhosphate pH 6.8 – 36.8 mL of 1 M Na2HPO4 + 38.2 mL of 1 M NaH2PO4
    250 mM NaCl – 25 mL of 5 M
    2X Denhardt’s – 10 mL of 100X
    10% PEG 8000 (6000) W/V – 50 g (add last after SDS is dissolved)
    ddH2O – up to 1 L

    Northern Wash A for 1L
    2X SSC – 100 mL of 20X
    1% SDS – 100 mL of 10%
    ddH2O – up to 1L

    Northern Wash B for 500 mL
    0.2X SSC – 5 mL of 20X
    0.1% SDS – 5 mL of 10%
    ddH2O – up to 500 mL

    Denaturing Mix for 100 uL
    ddH2O – 22.5 uL
    10X MOPS – 10 uL
    37% Formaldehyde – 17.5 uL
    Formamide – 50 uL