Troubleshooting guide

1. Low efficiency of transfection of cells by liposomes, electroporation and calcium phosphate methods. If cells are normal and the cell density is appropriate, it is likely that the amounts of DNA constructs used and parameters applied in transfection are not optimal. Make sure to optimize the conditions for transfection of the cells, including DNA concentration, lipids ratio or voltage pulse. In the case of stable transfection, ensure that the concentration of drug used in selection is not too high. A killing curve should be established.

2. Low titer of retroviruses. The virus stock is overdiluted or density of retrovirus particles is low due to low efficiency of stable transfection of packaging cells with modified virus DNA constructs. Try to use a good packaging cell line and optimize the parameters used for transfection. Reinfection may be performed to amplify the virus titer.

3. Low efficiency of transfection with frozen high-titer viruses. Assuming transfection conditions were optimal, it is very likely that the viruses lost significant activity. It is recommended that high-titer viruses be used in a couple of hours. Frozen aliquots should not be frozen and thawed.

4. No antisense RNA is expressed in transfected cells. This could be due to the orientation of the introduced cDNA. Make sure that the poly(A) strand of the cDNA is inserted downstream from the driving promoter in 3' to 5' orientation in order to make the antisense RNA or poly(T) strand. Try to check the mRNA level in the cells. If the level is equivalent to that of the control cells, the wrong orientation of the introduced cDNA is the case.

5. Low reduction of the protein of interest. This is obviously low efficiency of antisense inhibition. Try to run a northern blot analysis to check for antisense RNA expression using labeled sense RNA as a probe. Very little antisense RNA or not much reduction in mRNA level compared with nontransfected cells is exactly the cause of a low-percentage reduction of the protein. Additionally, it may be related to the activity of the promoter driving the expression of the introduced cDNA. If it is a constitutive promoter, its activity is low. Try to use a stronger promoter. If the driving promoter is inducible, try to optimize the concentration of the inducer.

6. Some of drug-selected stably transfected cell clones show 100% death during subsequent expanding or culture in a selection medium. If these clones are not frozen, it is certain that these isolated clones are false stably transfected cells. It is likely that during trypsinization and resuspension, the cells were not well suspended; instead, they aggregated. After transfection, some of cells within the aggregated colonies received a smaller amount of drug and still underwent proliferation, forming false colonies that were picked up. During the trypsinizing and picking-up, these cells were loosened by physical force. Because they were not particularly stable colonies, they were killed in drug selection medium during subsequent expanding or culture.


1. Wu, W. and Welsh, M.J., Expression of HSP27 correlates with resistance to metal toxicity in mouse embryonic stem cells transfected with sense or antisense HSP27 cDNA, Appl. Toxicol. Pharmacol., 141, 330, 1996.

2. Robert, L.S., Donaldson, P.A., Ladaigue, C., Altosaar, L., Arnison, P.G., and Fabi-janski, S.F., Antisense RNA inhibition of ß-glucuronidase gene expression in transgenic tobacco can be transiently overcome using a heat-inducible ß-glucuronidase gene construct, Biotechnology, 8, 459, 1990.

3. Rezaian, M.A., Skene, K.G.M., and Ellis, J.G., Antisense RNAs of cucumber mosaic virus in transgenic plants assessed for control of the virus, Plant Mol. Biol., 11, 463, 1988.

4. Hemenway, C., Fang, R.-X., Kaniewski, W.K., Chua, N.-H., and Tumer, N.E., Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA, EMBO J., 7, 1273, 1988.

5. Dag, A.G., Bejarano, E.R., Buck, K.W., Burrell, M., and Lichtenstein, C.P., Expression of an antisense viral gene in transgenic tobacco confers resistance to the DNA vu-us tomato golden mosaic virus, Proc. Natl. Acad. Sci. USA, 88, 6721, 1991.

6. Iwaki, T., Iwaki, A., Tateishi, J., and Goldman, J.E., Sense and antisense modification of glial aB-crystallin production results in alterations of stress fiber formation and thermoresistance, J. Cell Biol., 15, 1385, 1994.

7. Yang, N.S., Burkholder, J., Roberts, B., Martinell, B., and McCabe, D., In vitro and in vivo gene transfer to mammalian somatic cells by particle bombardment, Proc. Natl. Acad. Sci. USA, 87, 9568, 1990.

8. Wu, W., Gene transfer and expression in animals, in Handbook of Molecular and Cellular Methods in Biology and Medicine, Kaufman, P.B., Wu, W., Kim, D., and Cseke, L., CRC Press, Boca Raton, FL, 1995.

9. Zelenin, A.V., Titomirov, A.V., and Kolesnikov, V.A., Genetic transformation of mouse cultured cells with the help of high-velocity mechanical DNA injection, FEBS Lett., 244, 65, 1989.

10. Hasty, P. and Bradley, A., Gene targeting vectors for mammalian cells, in Gene Targeting: A Practical Approach, Joyner, A.L., Ed., IRL Press, Oxford University Press, Inc., New York, 1993.

Analysis of Gene Expression at the Functional Genomic Level



Principles and General Considerations Isolation of Total RNAs and Purification of mRNAs Electrophoresis of RNAs Using Formaldehyde Agarose Gels Blotting of RNAs onto Nylon Membranes by the Capillary Method Preparation of Isotopic or Nonisotopic DNA/RNA Probes Protocol A. Preparation of DNA Probes

Protocol B. Preparation of RNA Probes by Transcription in Vitro Hybridization and Detection of Signals

Analysis of mRNA Expression by a Semiquantitative PCR Approach

Troubleshooting Guide


Northern blot hybridization is a procedure in which different sizes of RNA molecules are separated in an agarose gel, immobilized onto a solid support of either nylon or nitrocellulose membranes, and then subjected to hybridization with a labeled DNA or RNA probe. Unlike the Southern blotting technique that involves DNA-DNA hybridization, northern blot hybridization refers to RNA-DNA or RNA-RNA hybridization. Technically, northern is not a person's name; nor is it due to a geographic location. As a matter of fact, this technique was developed later than the Southern blot method, and thus named the northern blot method.

The northern blot technique is one of the most fundamental and powerful tools used in analysis of gene expression.1 It is a sensitive, reliable and quantitative method widely employed in the characterization of the steady-state level of RNA transcripts. RNA analysis is a powerful approach to monitoring the activity of an endogenous or introduced gene in specific cell lines or tissues. In spite of the fact that other methods such as RNase protection and dot/slot blot hybridization can be used for


RNA analysis and are relatively more sensitive, the northern blot method has several advantages. As has been demonstrated previously,2-4 expression patterns of genes are often complex. Multiple RNA molecules can be expressed from a single gene and thousands of RNA species are generated from a single cell, tissue or organism. Northern blot analysis can simultaneously provide information on the species, sizes and expression levels of diversity of RNAs that cannot be displayed by alternative techniques such as the dot/slot blot and RNA protection assays. Also, membrane filters containing a record of different RNA species can be reused for analysis of multiple RNAs' transcripts from several genes in the same RNA sample. Therefore, the value and applications of northern blot technology are immeasurable.

This chapter offers detailed protocols for standard northern blot hybridization and for semiquantitative PCR and dot blot analysis of mRNA expression.1,2,4-9


The primary principles and procedures of northern blot hybridization are illustrated in Figure 10.1. Different RNAs are first denatured by formaldehyde and then subjected to separation according to their molecular weights by standard agarose gel electrophoresis. An agarose gel serves as a molecular sieve; an electron transport in an electrical field is the driving force for migration of negatively charged RNAs during electrophoresis. The separated RNAs are then blotted or transferred onto a nylon or nitrocellulose membrane. Generally, different positions of RNAs in the agarose gel are identically recorded on the membrane. After covalent cross-linking of RNA molecules on the membrane, specific or target sequences of RNAs can be hybridized with a DNA or RNA probe of interest, followed by detection with an appropriate method.

One important concept to know is that the principle of mRNA transcribed from its gene or DNA is the base pairing rule. That means that the sequence of an mRNA is complementary to that of the DNA template from which it was copied, except that base T is replaced by U. It is exactly the base pairing or complementary rule that makes it possible for a single-stranded DNA or RNA probe to hybridize with its target sequences of denatured mRNA by means of hydrogen bonds. With regard to probes, an RNA probe or riboprobe transcribed in vitro has been demonstrated to be more sensitive or hotter than a DNA probe.1 In addition, RNA-RNA hybrids are more stable compared with RNA-DNA hybrids. Therefore, hybridization and washing can be carried out under relatively high stringency conditions, thus reducing potential development of a nonspecific background.

Because RNAs, as compared to DNA, are very mobile molecules due to potential degradation by RNases, much care should be taken to maintain the purity and integrity of the RNA. This is certainly critical for northern blot hybridization. RNase activity is not readily inactivated. Two major sources of RNase contamination are from the user's hands and from bacteria and fungal molds present on airborne dust particles. To avoid RNase contamination, gloves should be worn and changed frequently when handling RNAs. Disposable plasticware should be autoclaved. Non-disposable glass- and plasticware should be treated with 0.1% diethyl pyrocarbonate (DEPC) in d.H2O and be autoclaved prior to use. DEPC is a strong RNase inhibitor.

Separation of RNAs on an agarose gel

Transfer of RNAs onto a membrane

Hybridization with a nonradioactive or a radioactive probe

Detection of hybridized signals

FIGURE 10.1 Scheme of mRNA analysis by northern blot hybridization.

Glassware may be baked at 250°C overnight. Gel apparatus should be soaked with 0.2% SDS overnight and thoroughly cleaned with detergent followed by thorough rinsing with DEPC-treated water. Apparatus for RNA electrophoresis, if possible, should be separated from that used for DNA or protein electrophoresis. Chemicals to be used should be of ultrapure grade and RNase-free. Gel mixtures, running buffers, hybridization solutions and washing solutions should be made with DEPC-treated water or treated with a few drops of DEPC.

Similar to Southern blot hybridization, a standard northern blot consists of five procedures: (1) preparation of RNAs to be analyzed; (2) agarose gel electrophoreis of RNA molecules; (3) blotting of RNAs onto a solid membrane; (4) hybridization of the RNA with a labeled DNA or RNA probe; and (5) detection of the hybridized signals. Agarose gel electrophoresis is a common tool for separating different RNAs. Agarose comes from seaweed algae and is a linear polymer basically composed of D-galactose and L-galactose. Once agarose powder is melted and then hardened, it forms a matrix that serves as a molecular sieve by which different sizes of RNAs are separated during electrophoresis.

A number of general factors should be considered when agarose gel electro-phoresis of RNA is performed: an ultrapure grade agarose that is RNase-free is highly recommended for northern blotting and the concentration of agarose will influence separation of RNAs. (A general chart is shown below.) Normally, the larger the size of the RNA is, the lower the percentage of agarose that should be used to obtain sharp bands.

During electrophoresis, RNAs move through the gel matrix at a rate inversely proportional to the log of their molecular weight. Small RNA species move faster than large RNA molecules.

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