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Invitrogen™ NorthernMax™ Formaldehyde Load Dye

Catalog No. AM8552
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AM8552 6 x 1 mL tubes
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Catalog No. AM8552 Supplier Invitrogen™ Supplier No. AM8552
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Description

Ready-to-use solution

  • To use, add solution to RNA sample (3 parts solution: 1 part sample) and heat briefly:The samples are then ready to be loaded
  • Ethidium bromide can be added to the samples if desired
  • Compatible with formaldehyde-containing gels
  • The same solution used in Invitrogen kits

Agarose Gel Electrophoresis, DNA and RNA Purification and Analysis, Northern Blotting, Nucleic Acid Gel Electrophoresis and Blotting

Order Info

Shipping Condition: Room Temperature

Specifications

Content And Storage Store at -20°C.
Gel Compatibility Formaldehyde-Containing Gels
Quantity 6 x 1 mL tubes
Shipping Condition Room Temperature
Product Type Northern Analysis Loading Dye

Frequently Asked Questions (FAQs)

Should I include ethidium bromide in my agarose gel when visualizing RNA?

You can add ethidium bromide to the sample loading dye or to the RNA samples to a final concentration of 10 µg/mL for direct visualization of the RNA during and after electrophoresis. Adding ethidium bromide to the samples or loading buffer is preferable to adding it to the gel or post-staining the gel, since formaldehyde in the gel will interact with the ethidium bromide, resulting in fluorescence, making it difficult to discern specific staining. Using 10 µg/mL ethidium bromide reportedly reduces assay sensitivity by 5-10%. However, the benefit of knowing that the RNA is intact, that the gel ran well, and that all of the RNA transferred, can be worth this minor loss in sensitivity.

How can I ensure that all of an RNA sample transfers from gel to membrane?

Incomplete transfer is often caused by short-circuiting. Strips of Parafilm sealing film around the outside edges of the gel can prevent this. Large RNA species may not transfer well because of their size. A basic transfer buffer (e.g., NorthernMax One-Hour Transfer Buffer) will partially shear the RNA so that larger RNA species transfer more efficiently. Check RNA transfer by including ethidium bromide in RNA samples or staining the gel in ethidium bromide after transfer and viewing your gel under UV light. RNA markers are invaluable to demonstrate whether large RNAs have fully transferred. Our Invitrogen Millennium Markers are especially useful for this purpose, since they include transcripts at 1,000 nt intervals from 0.5 to 9 kb.

I am seeing multiple bands after RNA hybridization, but I only expected one band. Why am I seeing cross-hybridization?

The following reasons could have led to cross-hybridization:

  • The probe concentration was too high.
  • Hybridization/washing conditions were not stringent enough.
  • There were multiple targets in the mRNA.
  • There was too much nonhomologous sequence in the probe.
  • Cross-hybridization to ribosomal bands. This can occur when the total RNA has a large amount of rRNA on the blot, trapping the probe. In this case, you should see the specific band, but it may be much fainter.
  • The hybridization temperature was too low (try increasing it up to 52°C).

    • I am seeing high background following RNA hybridization. What could be causing this?

    There are several types of background, and each can have a different cause:

    1) Blotchy signal across the membrane:
    This can be caused by a membrane of poor quality, one that has dried out, or one that has been mishandled (e.g., oil from human skin, powder from gloves). Use high quality nylon membrane that has not previously been handled and use forceps to handle the membrane from the edges. Blotchiness can also be caused by uneven distribution of the hybridization reagents. Do not pipette probe directly onto the membrane in hybridization solution; dilute it into the hybridization solution first.

    2) A smear through the lane:
    Hybridization conditions that are substantially below the optimum for a given probe can lead to high lane-specific background and/or substantial cross-hybridization. Start with a high hybridization temperature and slowly decrease the temperature until a specific signal is obtained. High probe concentrations, especially for nonisotopic probes, can also cause lane-specific background. Use 10 pM nonisotopically labeled DNA probes and 0.1 nM nonisotopically labeled RNA probes.

    3) Speckling across the membrane:
    Probe preparations with poor incorporation (or where unincorporated nucleotides have not been removed) can cause speckling on the membrane. Check probe quality and remove unincorporated nucleotides. Particulates in probe preparations or hybridization buffer (e.g., when not completely in solution) can also cause speckling on the membrane. Ensure that these reagents are in solution, and consider centrifuging in a microfuge or low-speed centrifuge, or filtering the solutions through a 0.22 µm filter to remove particulates.

    If you see high background that is not associated with the lanes, this could be due to:

  • A bad membrane or incompatible membrane.
  • A membrane that dried out during procedure.
  • Reagents that were not evenly distributed.
  • Microbial contamination.
  • Particulate matter deposited on the membrane.
  • Precipitates present in nonisotopic detection reagents.
  • Agarose or transfer buffer that dried on the membrane.
  • Static charges developing during film development.
  • A blot that was too wet when exposed to film.

    • I am getting a very poor signal following RNA hybridization. What could be the cause of this?

    Poor signal could be a result of the following:

    - A hybridization temperature that was not optimal.
    - Probe degradation (too old).
    - A low specific activity probe (should be ~2 x 10^9 cpm/ug, random primed).
    - A probe that was not denatured (DNA).
    - A probe concentration that was too low (<10^6 cpm/mL).
    - A longer hybridization time needed.
    - Poor transfer of RNA to membrane.
    - Inadequate cross-linking or overexposure to UV light.
    - An alkaline transfer time that was too long (>4 hours).
    - The wrong membrane (nitrocellulose).
    - Failure to follow nonisotopic detection protocols.
    - A message that co-migrates with ribosomal RNA.
    - Inappropriate use of intensifying screens.

    I am seeing residual RNA in my gel following transfer. What could be the cause of this?

    Residual RNA could be due to:

    - An inadequate volume of transfer buffer (>0.5 mL/cm2)
    - Too much weight
    - The gel used was too thick (>6 mm)
    - The transfer time was insufficient (we recommend 15 min per mm of gel thickness)

    How can I prevent cross-hybridization to rRNA sequences?

    rRNA makes up ~80% of total RNA samples. When 10 µg of total RNA is loaded into a Northern gel lane, the 18S and 28S rRNA bands contain 2-6 µg RNA each. This amount of nucleic acid can nonspecifically trap probe as well as bind complementary sequence. Probe trapping by rRNA can be reduced by using the minimal amount of probe, and by labeling only sequence complementary to mRNA. Transfer using a basic buffer can prevent trapping. Finally, you can use a high hybridization and wash temperature to minimize cross hybridization to rRNA.

    Can I reprobe my Northern blot? Do you offer stripping recommendations?

    For RNA probes on DNA or RNA targets:
    Autoclave the membrane in a bottle containing 0.1% SDS solution for 15 minutes. Repeat if necessary.

    For DNA probes on DNA targets only:
    You can use the same protocol used for RNA probe stripping.

    Another option is alkaline denaturation. Incubate the membrane with 400 mM NaOH for 30 minutes, then wash with 0.1% SDS for 15 minutes. These stripping methods should work for 2 to 3 stripping procedures. However, nucleic acids will gradually be removed from the blot.
    How can I increase the sensitivity of my Northern hybridizations?

    Please see below the top ten ways to increase sensitivity of your Northern hybridizations:

    1) Increase the amount of RNA loaded in each lane (up to 30 mg).
    2) Use poly(A) RNA instead of total RNA; 10 mg of poly(A) RNA is ~300-350 mg total RNA (3-5%).
    3) Switch to ULTRAhyb Ultrasensitive Hybridization Buffer.
    4) Switch from DNA to RNA probes.
    5) Use downward alkaline capillary transfer.
    6) Use an optimal hybridization temperature.
    7) Use a freshly synthesized probe.
    8) Use a high specific activity probe (10^8 to 10^9 cpm/mg).
    9) Increase exposure time (it can take up to 3 days to see low-abundance messages with radiolabeled probes).
    10) Follow the manufacturer's recommendations to crosslink the RNA to the membrane.

    Read more about these suggestions here (https://www.thermofisher.com/us/en/home/references/Invitrogen-tech-support/northern-analysis/general-articles/ten-ways-to-increase-the-sensitivity-of-northern-hybridizations.html).

    Is it possible to make the Northern blotting procedure faster?

    Running small 10 cm gels for Northern blotting takes 30-90 minutes, much quicker than larger gels. The biggest time savings, however, can be during transfer to the membrane. Traditionally, Northerns have been blotted overnight using capillary transfer and a high-salt buffer (10X SSC or 10X SSPE). By using a weak base as the medium (e.g., NorthernMax One-Hour Transfer Buffer), the transfer can be completed in just 1 hour. Alternatively, you can electroblot your RNA in 1 hour.

    Can Northern blotting be used to detect small RNAs?

    Yes. You can use a 15% denaturing polyacrylamide gel for Northern analysis of small RNAs, such as miRNAs and siRNAs. A hyridization buffer optimized for use with short probes, such as ULTRAhyb-Oligo solution, should be used for the best results. For more sensitive detection, enrich the RNA sample for small RNAs (e.g., with the mirVana miRNA Isolation Kit or mirVana PARIS RNA and Native Protein Purification Kit. Of course, using a solution hybridization assay such as the mirVana miRNA Detection Kit to analyze small RNAs can provide much greater sensitivity and allow you to detect multiple small RNAs simultaneously.

    Are there differences between traditional and miRNA Northern blotting?

    We recommend following the instructions in our mirVana miRNA Isolation Kit (Cat. No. AM1560, AM1561) for miRNA Northern blotting. We recommend starting with 2 µg total RNA or a 1 µg miRNA-enriched sample. Run your samples on a denaturing polyacrylamide gel, as the RNA may be too small to detect on an agarose gel. Transfer RNA to a nylon membraneby electroblotting. The compositions of recommended prehybridization, hybridization, and wash buffers are provided in the manual.

    Do you have suggestions for transfer and fixation of RNA to a positively charged nylon membrane?

    These are our suggestions:

    - Because alkaline transfer can overhydrolyze small RNAs, and hence decrease their binding, do not exceed 4 hours of transfer.
    - Pour gels as thin as possible (usually between 5-6 mm).
    - We recommend 15-20 minutes of transfer time per millimeter of gel thickness.
    - Crosslinking (using UV) or baking (100°C for 10 minutes) is essential to assure that nucleic acids are irreversibly bound to the membrane.

    Why run a glyoxal gel instead of a formaldehyde denaturing agarose gel?

    Using glyoxal/DMSO instead of formaldehyde avoids the need to pour and run gels in a fume hood and eliminates safety issues associated with formaldehyde.

    For Research Use Only. Not for use in diagnostic procedures.